Lab 1 Buffer and Solution Preparation

Buffers

All weak acids and weak bases have a balance reaction in aqueous forms. They donate/accept H+
ions to/from water and form their conjugate base/acid forms according to their reaction balance.

HA A- + H+

As it is shown above, the HA weak acid interacts with water and forms its conjugate base A-

Buffers are the aqueous solutions that include a weak acid and its conjugate base or a weak base
and its conjugate acid and provide toleration to pH changes in a definite range. This property of buffers
provides keep the pH majorly constant during the chemical applications. All buffers have a buffer
capacity as a result of the balance reaction of conjugate acid/base couples.

According to Le Chatelier’s principle, when a strong base is added to a buffer, H+ ions of conjugate
acid reacts with OH- ions of the strong base and balance of the reaction shifts to the basic side slightly.
For example, in the balance reaction below; H+ ions of the HA conjugate acid reacts with the OH- group
of the strong base and forms more A- ions.

HA + OH- A- + H2O

On the other hand, when a strong acid is added to a buffer system; ions behave vice versa and the
balance of the reaction shifts to the acidic side.

In this example, if it is continued to add strong base/acid to the buffer system, pH changes slightly
until the buffering capacity is exceeded. Buffer capacity is a pH range (pKa ± 1) which defines the
resistance of the buffer [1 ].

Many biochemical reactions in biological systems need a definite pH to occur or show maximal
activity. Therefore, there are many biochemical products which have an acid/base property and many
natural fluids having a buffering capacity. Blood is a very good example for these biological buffers. In
addition, there are many buffer systems that are suitable for in vitro studies such as phosphate buffer
saline (PBS).

Buffer Calculations and Preparation

There are 3 ways to prepare a buffer solution:

1- Using Handerson-Hasselbalch Equation

Handerson-Hasselbalch equation is a commonly used formula which is derivated from the

acid/base balance reaction.

1
 HA A- + H+

According to the balance constant;

K = [H+][A-] / [HA]

- logK = - log[H+] - log[A-] /[HA]

pK = pH – log[A-]/[HA]

pH= pK + log[A-]/[HA]

pH= pK + log [conjugate base] / [acid]

2- Weak acid / strong base

Amount of the weak acid is calculated from the concentration of the buffer solution and pH is
adjusted with a pH meter by titration with a strong base.

3- Weak base / strong acid

Amount of the weak base is calculated from the concentration of the buffer solution and pH is
adjusted with a pH meter by titration with a strong acid.

Solutions

Biological systems also require many ions and molecules at definite concentrations for accessing
regularly. Concentration of a solution defines the amount of ions or molecules in unit of volume.

In analytical chemistry, there are some universal terms defining the concentration of the solution.

• Molarity (M) = n / V (mole/liter)

• Normality (N) = M / feq (molar concentration / equivalence factor)
• Molality (m) = n / msolvent (mole/ mass of solvent)
• Percentage (%) : mass / total volume or volume / total volume
• Mole or mass ratio : ppm, ppb, ppt (parts per million/billion/trillion)

Simple and serial dilution

Dilution is a technique to decrease the concentration of a solution by adding solvent and

increasing the total volume of the solution.

Dilution ratio can be calculated easily by the formula below;

C1. V1 = C2 . V2

2
 C1 : first concentration

V1: first volume

C2: last concentration

V2: last volume

Simple dilution is a one-step technique that a major stock is used only.

8X → X

Instead of simple dilution, serial dilution is a multi-step technique in which solutions are diluted
from the previous stocks.

8X → 4X → 2X → X

EXPERIMENTS
• Each group will prepare 0.1%-2%-2.5%-3% SDS Solutions (1mL)
• 1st group will prepare Z buffer (pH 4 and 5) by citric acid (50 ml) and 25 mM glucose (10 ml)
• 2nd group will prepare Z buffer (pH 6 and 8) by phosphate (50 ml) and 50 mM glucose (10 ml)
• 3rd group will prepare Z buffer (pH 9 and 10) by TRIS/HCl (50 ml) and 75 mM glucose (10 ml)
• 4th group will prepare 0,1 M pH7 phosphate buffered saline (PBS) by using Handerson-Hasselbalch
equation (250 ml) and 10mM glucose (10mL)

Z buffer per 50 ml :

0,06 M Na2HPO4 Use phosphate, citric acid or TRIS according to pH

0,04 M NaH2PO4

0,01 M KCl
0,001M MgSO4

Phosphate Buffered Saline (PBS)

0,137 M NaCl
0,027 M KCl
0,01 M NaH2PO4
0,0018 M Na2HPO4

[1] Basic principles of electrolyte chemistry for microfluidic electro Part I: Acid–base equilibria and pH buffers

Alexandre Persat, Robert D. Chambers and Juan G. Santiago, 2009

3
 Lab 2 Spectrophotometric Measurements
Spectrophotometry

Spectrophotometry is one of the most common techniques in biochemistry to determine or

quantify the samples easily. This technique is a type of spectrometry based on absorption or reflection
of light in visible-ultraviolet or near-infrared wavelength.

purple blue green yellow orange red

UV IR
~ 390 nm ~750 nm
Visible

Fig 1. Simple scheme of wavelength spectrum

When the light in a specific wavelength is passed through a solution, the intensity of the light
reduces as a result of absorption. Amount of the light that is soured by the material in solution refers
to the absorbance (A).

I0 I A= log (I0 / I)
Detector I0 : amount of the
ppp00000000000000000000000000000000
00000000000
Light source Cuvette light passing throughthe sample
l
I : amount of the light leaving the sample

Fig 2. of the spectrophotometric measurement

Lambert-Beer Law

Spectrophotometry is a useful technique to measure the amount of a material in a solution.

According to Lambert-Beer Law, there is a direct correlation between the absorbance (A) and the
concentration of the material (C) in a solution and the length of the light way ( l ). This correlation is
expressed by a constant number which is called extinction coefficient ().

A = . C. l

There are two exceptions that make the Lambert-Beer law invalid. If the concentration of the
sample is too high, the molecules in the solution may be interfered with the other molecules and the
absorbance is not detected correctly. When the absorbance value is higher than 2, this result is
accepted as unreliable.
4
 Secondly, if the light way is too long, the light will be absorbed more than it has to be. In other
words, all light can be absorbed and cannot be detected. To prevent this problem, most of the cuvettes
have 1 cm length.

Key points in spectrophotometric measurement

In spectrophotometric measurement there are some key points to be considered. First, you
should be sure that you measure the sample at right wavelength. If the wavelength of the sample gives
maximum absorbance is not known, sample should be scanned in a definite spectrum range.

absorbance Fig 3. Schematic show of the spectrum

scanning

200 700
wavelength

Second, you should use the right type of cuvette for spectrophotometric measurement. For
example, UV measurements require quartz cuvettes.

Third, there should not be any fleck or finger print on cuvette, air bubble or turbidity in sample
solution. It should be homogeny and clear in order to transmit the light.

Finally, concentration should be adjusted to a valid value for Lambert-Beer law.

SIMPLE DILUTIONS
1 M CuSO4 (700 nm) (total volume: 1ml)
Dilutions Stock (ml) dH2O (ml) Absorbance
1:2

1:5
1:10
1:20
1:50
1:100
1:200
unknown

5
SERIAL DILUTIONS
1 M CuSO4 (700 nm) (total volume: 1,5 ml)
Dilutions Stock (ml) dH2O (ml) Absorbance
1:3

1:9
1:27
1:81
1:243
1:729

Spectrum scanning

Scan the 1M CoCl2 solution from 200 nm to 800 nm and find the wavelength at which the cobalt
solution give maximum absorbance.

6
 Lab 3 Aminoacid Titration
Aminoacids

Amino acid is a biological molecule containing an amine group, a carboxyl group, a hydrogen
atom and a side chain connected to carbon atom in the center.

With these four different groups connected to the tetrahedral -carbon atom, amino acids have
two mirror-image forms are called the L isomer and the D isomer.

H R R H
H
+
H3N C
C C COO-
R
NH3+ NH3+
COO- C-terminus
COO- N-terminus
L-isomer D-isomer

Amino acids are found in nature in many types related to their side chains. But 22 of them are found
in proteins. In contrast to 20 of them which are encoded by the universal genetic code, pyrrolysine and
selenocysteine are incorporated into proteins by unique synthetic mechanisms.

Structures of 20 common amino acids [2]

Aliphatic R Groups

Aromatic R Groups

7
 Sulfur Containing R Groups Side Chains with Alcohol Groups

Basic R Groups Acidic R Groups and Their Amide Derivatives

Titration curves of the amino acids

Amino acids without charged groups on their side chains exist in neutral solution as zwitterions with
no net charge. A zwitterion has equal positive and negative charges; in other words, in solutionit is
electrically neutral.

When an aminoacid is titrated, its titration curve indicates the reaction of each functional group
with hydrogen ion. Each amino acid has characteristic values of Ka, pKa and pI of their titratable groups.
pI, isoelectric point is the pH at which the molecule has no net charge and in an electric field the
molecule does not migrate at its pI. This property is used in several separation methods.

pKa Values of Aminoacids

Amino acid Carboxyl Amino Amino acid Carboxyl Amino Side chain
group group group group
Glycine (G) 2,4 9,8 Threonine (T) 2,1 9,1
Alanine (A) 2,4 9,9 Cysteine (C) 1,9 10,7 8,4
Valine (V) 2,3 9,7 Tyrosine (Y) 2,2 9,2 10,5
Leucine (L) 2,3 9,7 Asparagine (N) 2,1 8,7
Isoleucine (I) 2,3 9,8 Glutamine (Q) 2,2 9,1
Methionine (M) 2,1 9,3 Aspartic acid (D) 2,0 9,9 3,9
Proline (P) 2,0 10,6 Glutamic acid (E) 2,1 9,5 4,1
Phenylalanine (F) 2,2 9,3 Lysine (K) 2,2 9,1 10,5
Tryptophan (W) 2,5 9,4 Arginine (R) 1,8 9,0 12,5
Serine (S) 2,2 9,2 Histidine (H) 1,8 9,3 6,0

8
 Amino acid A (0,05M)

0,05 M 0,05 M 0,05 M 0,05 M

NaOH pH NaOH pH NaOH pH NaOH pH
(ml) (ml) (ml) (ml)

1 26 51 76

2 27 52 77
3 28 53 78
4 29 54 79
5 30 55 80
6 31 56 81
7 32 57 82
8 33 58 83
9 34 59 84
10 35 60 85
11 36 61 86
12 37 62 87
13 38 63 88
14 39 64 89
15 40 65 90
16 41 66 91
17 42 67 92
18 43 68 93
19 44 69 94
20 45 70 95
21 46 71 96
22 47 72 97
23 48 73 98
24 49 74 99
25 50 75

[2] Principles of Biochemistry, Chapter 3, 57-61, Robert A. Horton, Laurence A. Moran, Gray Scrimgeour, Marc Perry, David
Rawn

9
Lab 4 Quantification of Proteins
Protein Determination by UV Absorption

1. Near UV Absorbance (280 nm)

Quantitation of the amount of protein in a solution is possible in a simple spectrometer. Absorption

of radiation in the near UV by proteins depends on the Tyr and Trp content. Therefore the A280 varies
greatly between different proteins. The advantages of this method are that it is simple, and the sample
is recoverable. The method has some disadvantages, including interference from other chromophores,
and the specific absorption value for a given protein must be determined. The extinction of nucleic
acid in the 280-nm region may be as much as 10 times that of protein at their same wavelength, and
hence, a few percent of nucleic acid can greatly influence the absorption.

Far UV Absorbance

The peptide bond absorbs strongly in the far UV with a maximum at about 190 nm. This very strong
absorption of proteins at these wavelengths has been used in protein determination. Because of the
difficulties caused by absorption by oxygen and the low output of conventional spectrophotometers
at this wavelength, measurements are more conveniently made at 205 nm, where the absorbance is
about half that at 190 nm. The advantages of this method include simplicityand sensitivity. Also the
sample is recoverable and in addition there is little variation in response between different proteins,
permitting near-absolute determination of protein. Disadvantages of thismethod include the necessity
for accurate calibration of the spectrophotometer in the far UV. Many buffers and other components,
such as heme or pyridoxal groups, absorb strongly in this region.

2. The Biuret Method for Protein Quantitation

2+
Cupric ions (Cu ) in the biuret reagent complex with the groups are involved in the peptide bond.

In the presence of alkaline media i.e. 3% NaOH and at least two peptide bonds a violet colored
2+
chelate is formed. Biuret also contains sodium potassium tartrate which assists in Cu complex
formation and further prevents their precipitation in an alkaline solution. The absorbance of the
colored chelate is measured at 540nm. The color intensifies from pink to reddish violet due to the
complexity of the peptide bond in the protein. The major advantage of this technique is that there is
no interference from materials that adsorb at lower wavelengths, and the technique is less sensitive
to protein type because it utilizes absorption involving peptide bonds that are common to all proteins,
rather than specific side groups. However, it has a relatively low sensitivity compared to other UV-
visible methods.

3. The Lowry Method for Protein Quantitation

The most accurate method of determining protein concentration is probably acid hydrolysis
followed by amino acid analysis. Most other methods are sensitive to the amino acid composition of
the protein, and absolute concentrations cannot be obtained. The procedure of Lowry et al. is no
exception, but its sensitivity is moderately constant from protein to protein, and it has been so
10
widely used that Lowry protein estimations are a completely acceptable alternative to a rigorous
absolute determination in almost all circumstances in which protein mixtures or crude extracts are
involved.
The method is based on both the Biuret reaction, in which the peptide bonds of proteins react with
copper under alkaline conditions to produce Cu+, which reacts with the Folin reagent, and the Folin–
Ciocalteau reaction, which is poorly understood but in essence phosphomolybdotungstate is reduced
to heteropolymolybdenum blue by the copper-catalyzed oxidation of aromatic amino acids. The
reactions result in a strong blue color, which depends partly on the tyrosine and tryptophan content.
The method is sensitive down to about 0.01 mg of protein/mL, and is best used on solutions with
concentrations in the range 0.01–1.0 mg/mL of protein.

4. The Bicinchoninic Acid (BCA) Assay for Protein Quantitation

The bicinchoninic acid (BCA) assay is similar to the Lowry assay, since it also depends on the
conversion of Cu2+ to Cu+ under alkaline conditions. The Cu+ is then detected by reaction with BCA.
The two assays are of similar sensitivity, but since BCA is stable under alkali conditions, this assay has
the advantage that it can be carried out as a one-step process compared to the two steps needed in
the Lowry assay. The reaction results in the development of an intense purple color with an absorbance
maximum at 562 nm. Since the production of Cu+ in this assay is a function of protein concentration
and incubation time, the protein content of unknown samples may be determined
spectrophotometrically by comparison with known protein standards. A further advantage of the BCA
assay is that it is generally more tolerant to the presence of compounds that interfere with the Lowry
assay. In particular it is not affected by a range of detergents and denaturing agents such as urea and
guanidinium chloride, although it is more sensitive to the presence of reducing sugars. Both a standard
assay (0.1–1.0 mg protein/mL) and a microassay (0.5–10 μg protein/mL) are described.

5. The Bradford Method for Protein Quantitation

A rapid and accurate method for the estimation of protein concentration is essential in many
fields of protein study. This technique is simpler, faster, and more sensitive than the Lowry method.
Moreover, when compared with the Lowry method, it is subject to less interference by common
reagents and nonprotein components of biological samples. The Bradford assay relies on the binding
of the dye Coomassie Blue G250 to protein. Detailed studies indicate that the free dye can exist in four
different ionic forms for which the pKa values are 1.15, 1.82, and 12.4. Of the three charged forms of
the dye that predominate in the acidic assay reagent solution, the more cationic red and green forms
have absorbance maxima at 470 nm and 650 nm, respectively. In contrast, the more anionic blue form
of the dye, which binds to protein, has an absorbance maximum at 590 nm. Thus, the quantity of
protein can be estimated by determining the amount of dye in the blue ionic form. This is usually
achieved by measuring the absorbance of the solution at 595 nm. The dye appears to bind most readily
to arginyl and lysyl residues of proteins. This specificity can lead to variation in the response of the
assay to different proteins, which is the main drawback of the method. The original Bradford assay
shows large variation in response between different proteins. Several modificationsto the method
have been developed to overcome this problem. However, these changes generally result in a less
robust assay that is often more susceptible to interference by other chemicals.

11
Consequently, the original method devised by Bradford remains the most convenient and widely used
formulation. The standard assay is suitable for measuring between 10 and 100 μg of protein.

The Protein Protocols Handbook,John M. Walker, 2002

Materials and Methods:

Protein expression

The genes encoding the Pseudomonas sp. esterase, PE were cloned into the vectors pET-24a (Novagen
Inc.). These clone were transformed into E. coli BL21(DE3) (Stratagene). The bacteria were grown in 50
ml LB medium in a 250 ml Erlenmeyer flask, supplemented with the appropriate antibiotic (25 µg/mL
kanamycine) , with 200 rpm shaking at 37 °C until OD600 = 0.6–1.0. Expression of the recombinant
protein was induced with 1 mM IPTG. After incubating an additional 3 h, cells were harvested by
centrifugation (4000 x rpm, 30 min, 4 °C). Cell pellets were washed with PBS, centrifuged, and
resuspended in 2.0 ml lysis buffer (0.5 M NaCl, 20 mM NanHnPO4, 5 mM imidazole, pH 7.4) for each
pellet derived from 50.0 ml cell culture.

Disruption of cells

E. coli cells expressing PE-(His)6 were suspended 1:2 (w/v) in 20 mM sodium phosphate, pH 7.4, 5 mM
imidazole, containing 0.5 M NaCl and disrupted by sonication (4 x 30 s on ice). The cell lysates were
centrifuged for 30 min at 15 000 rpm (Sorvall SS-34 rotor), and the supernatants were filtered and used
for purification of the His tagged protein.

Bradford Protein Assay

Prepare 5µg, 10 µg, 20 µg, 40 µg, 50 µg, 75 µg, 100 µg BSA solutions as standard solutions to draw
standard curve. Add 5ml Bradford reagent, wait 15min. and read absorbance at 595nm.

Read absorbance of the sample using the same protocol and find the protein concentration.

12
Lab 5. Column Chromatography
In column chromatography, the stationary phase, a solid adsorbent, is placed in a vertical glass
(usually) column and the mobile phase, a liquid, is added to the top and flows down through the
column (by either gravity or external pressure). Column chromatography is generally used as a
purification technique: it isolates desired compounds from a mixture.

Types of column chromatography:

• Ion Exchange (protein charge)

• Size Exclusion (separates on size)

• Hydrophobic Interaction (hydrophobicity)

• Affinity:

• Protein A

• His-tagged

• Glutathione-s-transferase

AFFINITY CHROMATOGRAPHY

Affinity chromatography is a method of separating biochemical mixtures based on a highly specific

interaction.

Affinity chromatography can be used to:

• Purify and concentrate a substance from a mixture into a buffering solution

• Reduce the amount of a substance in a mixture
• Discern what biological compounds bind to a particular substance
• Purify and concentrate an enzyme solution.

In affinity chromatography, the immobile phase is typically a gel matrix (usually agarose). the
starting point is an undefined heterogeneous group of molecules in solution, such as a cell lysate,
growth medium or blood serum. The molecule of interest will have a well known and defined property
which can be exploited during the affinity purification process. The process itself can be thought of as
an entrapment, with the target molecule becoming trapped on a solid or stationary phase or medium.
The other molecules in solution will not become trapped as they do not possess this property. The solid
medium can then be removed from the mixture, washed and the target molecule released from the
entrapment in a process known as elution.

Ni-Affinity Chromatography

Ni-Affinity Chromatography uses the ability of His to bind nickel. Six histadine amino acids at the
end of a protein (either N or C terminus) is known as a 6X His tag. Nickel is bound to an agarose bead
by chelation using nitroloacetic acid (NTA) beads. The general method is to batch absorb the protein
onto the column, by mixing the beads with the sample, then pouring the slurry of NTA beads and

13
protein into a column, where low concentrations of phosphate and imidazole are used to remove low
affinity bound proteins. If needed, the imdazole can be increased to 20 mM before most His tagged
proteins are eluted. Finally, higher concentrations of imidazole is used to elute the protein from the
NTA-beads.

His tag and column interaction

• His tags are typically a series of 6 histidines added to the C or N terminus of a recombinant
protein

Imidazole Histidine

• His and imidazole structure similarities

• Imidazole competes with His for Ni2+ sites

STEPS:

• Pour column

• Wash resin to remove packing buffer

• Equilibrate resin

• Bind His-tag protein

• Elute unbound proteins

• Wash protein bound onto the resin

• Elute His-tag protein

METHODS

See Sigma-Aldrich HIS-Select Nickel Affinity Gel Technical Bulletin.

14
Lab 6 Size Exclusion Chromatography
Terms in chromatography

The eluate is the mobile phase leaving the column.

The eluent is the solvent that will carry the analyte.
An immobilized phase is a stationary phase which is immobilized on the support particles, or on the
inner wall of the column tubing.
The mobile phase is the phase which moves in a definite direction. It may be a liquid (LC and CEC), a
gas (GC), or a supercritical fluid (supercritical-fluid chromatography, SFC). The mobile phase consists
of the sample being separated/analyzed and the solvent that moves the sample through the column.
Preparative chromatography is used to purify sufficient quantities of a substance for further use,
rather than analysis.
The retention time is the characteristic time it takes for a particular analyte to pass through the system
(from the column inlet to the detector) under set conditions.
The stationary phase is the substance which is fixed in place for the chromatography procedure.
Examples include the silica layer in thin layer chromatography.
The void volume, Vo represents the volume outside of the beads. Vo is determined by using a very
large molecule that is larger than the exclusion range for the gel.
The elution volume, Ve is actual volume that it requires to "elute" a biomolecule from a SEC column

Size exclusion chromatography Theory

Size exclusion chromatography is also often called molecular sieve, gel permeation or gel filtration
chromatography. This analytical method separates molecules based on their molecular sizes and
shapes by using the molecular sieve properties of a variety of porous materials. Molecular mixtures
separate on a size exclusion column because smaller molecules spend more time (travel a longer path)
going through the column. The molecules that are small enough to enter the holes of the whiffle balls
bounce around inside the balls and take a while to escape. Molecules bigger than the holes just flow
around the whiffle balls and thus travel a shorter, faster path in going through thecolumn.

15
 This resin is the SEC stationary phase. The resin used is based on type and size of the molecule(s) to
be separated and the degree of resolution required. The resolution depends on particle size, pore size,
flow rate, column length, sample volume and the mobile phase. Mobile phase is the solvent thatis used
to elute the molecules to be seperated. In size exclusion chromatography a buffer with constant pH
with at least 100mM NaCl concentration is used. The salt is necessary to reduce the interaction
between the proteins and the beads.

Size exclusion chromatography is used to separate and analyze a variety of chemicals, including
both synthetic polymers and biopolymers. It is a powerful and popular method to purify and determine
the molecular weight of proteins.

The elution volume (Ve) decreases roughly linear with the logarithm of the molecular
hydrodynamic volume. Columns are often calibrated using 4-5 standard samples (e.g., folded proteins
of known molecular weight), and a sample containing a very large molecule such as thyroglobulin to
determine the void volume. The elution volumes of the standards are divided by the elution volume of
the thyroglobulin (Ve/Vo) and plotted against the log of the standards' molecular weights.

16
 General Protocol for SEC

1. Prepare elution buffer which is 200mM NaCl 100mM Phosphate buffer pH 7.

2. Take required amount of bead powder and let it to swell (using required amount of elution
buffer) for at least 1 h. (1g bead G75 bead swells to 10-12ml).
3. Prepare a chromatography column with fitting and pour the swelled beads to column and
equilibrate the column washing the column with 3 volume elution buffer.
4. Load the column with your sample with blue dextran and orange G dye.
5. Wash with 1 volume of elution buffer and start collecting when blue dextran is at bottom of
the column, collect 4 droplets of fractions.
6. Continue to collect fractions until the orange G has run through the column. Continue to
collect for at least another 10 ml.
7. Analyze each tube for the protein for total protein concentration (Bradford assay)

Sample1 dye mixture with orange G, blue dextran and CoCl2.

Sample2 is BSA, lysozyme mixture.

17
Lab 7 SDS-PAGE
Electrophoresis is a very common technique to discriminate the biological molecules, determine
their sizes and analyze their purity. The main principle of electrophoresis is running molecules in the
presence of electric field and separating them by the help of a supporting material. This supporting
material can be cellulose for low-molecular weight samples such as amino acids or carbohydrates. On
the other hand, agarose and polyacrylamide gels are more useful for high-molecular weight samples.
Direction of technique (vertical or horizontal), thickness of the gel, buffers, dyes, applications etc.
also varies according to type of the technique and supporting material.

Polyacrylamide is a porous system consists of cross-linked acrylamide and bisacrylamide molecules.

Fig1. Copolymerization reaction of acrylamide and N,N’-methylene bisacrylamide [1]

Polyacrylamide gel electrophoresis (PAGE) is an advantageous technique that it has a high

resolution for small molecules and proteins/nucleic acids with differentiated sizes. Furthermore,
physically and chemically stable gel phase prevents the interaction of sample molecules with the gel
structure during migration. It is also possible to moderate the size of pores by adjusting the acrylamide-
bisacrylamide concentrations and modify the system according to the type of sample. As well as the
acrylamide-bisacrylamide ratio increases, pore sizes get smaller as a result of high number of cross-
links.

In proteomic analyses, in addition to native type, polyacrylamide gel electrophoresis can be

combined with a negatively charged denaturing detergent called sodium dodecyl sulfate (SDS).

18
 SDS breaks the all weak interactions of amino acids, thus protein is denatured and negatively
charged single polypeptide chains are formed. In order to break the disulfide bonds, SDS is not strong
enough; therefore samples are boiled in the presence of -mercaptoethanol before loading to the gel.

SDS + 2-mercaptoethanol

In consequence, negatively charged peptide chains migrate from cathode to anode, in another words
from negative to positive electrodes.

Contents of the SDS gel and their functions

• Acrylamide-bisacrylamide mix: Main components of polyacrylamide that provide the

porous gel structure
• TRIS/HCl buffer: Keeps the pH stabile in both stacking and resolving gels, also Cl- ions are
necessary for mobility (pH 6,8 in stacking gel; pH 8,8 in resolving gel)
• SDS: Denatures the protein structure and charges them negatively
• APS (Ammonium persulfate): Initiates the polyacrylamide formation reaction
• TEMED (Tetramethylethylene diamine): Catalyze this polymerization reaction

Other necessary buffers

Running Buffer

• Glycine: Facilitates the migration of molecules via sandwich system

• Tris: Stabilize the pH and these ions have electrolyte effect that complete the circuit
system
• SDS

Sample buffer

• pH 6,8 Tris/HCl buffer

• Glycerol: Precipitates in the wells with the samples as a result of its viscous character
• 2-mercaptoethanol: In 95oC breaks the disulfide bridges and all subunits of proteins are
separated from each other
• SDS
• Bromophenol blue: Blue colored dye is useful for visualizing the migration. It has a slight
negative charge and runs faster than all samples. It is easy to observe the end of
experiment by the help of this dye.

19
 SDS-PAGE includes two different gel layers; stacking gel and resolving gel. Ionic strength, pH and
pore size of these gels are different from each other and by this way they provide the effective
separation.

Top layer, stacking gel includes 5% acrylamide-bisacrylamide mix. Therefore the pores are large,
they facilitate the mobility. During the protein/nucleic acid samples are running through the stacking
gel, a sandwich system occurs. Chloride ions are negatively charged and they move faster than the
other molecules. Preparation of stacking gel requires TRIS-HCl pH 6,8 and in this pH glycine amino acid
exists in zwitterion form which has no net charge. Therefore negatively charged larger molecules take
place between chloride ions and neutral glycine aminoacid.

Cl- > protein/nucleic acid > Gly

This sandwich mechanism is necessary for stacking of samples in the same line.

On the other side; bottom layer resolving gel includes min 8% , max 15-20% acrylamide mix. This
means that resolving gel has smaller pores than stacking gel. pH of this gel is 8,8 so glycine turns to
its negatively charged form from zwitterion. Chloride and glycine ions move faster than proteins or
nucleic acids as a result of their smaller sizes. They carry the current and facilitate the mobility. As a
result of smaller pore sizes, smaller proteins /nucleic acids run faster than the larger molecules.

Cathode (-)
Stacking
gel

Resolving
gel

Anode (+)

[1] Rodney Boyer, Modern Experimental Biochemistry, Third Edition, Chapter 4; Characterization of Proteins and Nucleic Acids by
Electrophoresis

20
 Experimental Procedure

1st layer (bottom) (5 ml) 2nd layer (resolving) 3rd layer (stacking)
(12%, 15 ml) (5%, 5ml)
H2O 1,1 4,9 3,4
30% acrylamide mix 2,5 6,0 0,83
1,5 M Tris (pH8,8) 1,3 3,8 -
1,0 M Tris (pH 6,8) - - 0,63
10% SDS 0,05 0,15 0,05
10% ammonium persulfate 0,05 0,15 0,05
TEMED 0,005 0,006 0,005

Use max 1-2 ml 1st layer to prevent the leaking of resolving gel

Samples are mixed with sample buffer (1:1) and boiled 5 min.

After the gel is polymerized, system is completed. Running buffer is added and then samples are
loaded to each well.

Stacking gel: 45 V ~30 min

Resolving gel: 90 V ~1 hour

Take the gel and keep in staining solution overnight.

21
Lab 11 Enzyme Kinetics
General Properties of Enzymes

The greatest majority the biochemical reactions in living organisms do not take place spontaneously.
Biochemical reactions are catalized by enzymes which accelerates chemical reaction. Without
enzymes, these reactions take place at a rate far too slow for the pace of metabolism. All known
enzymes are proteins. Enzymes can be denatured and precipitated with salts, solvents and other
reagents. Many enzymes require the presence of other compounds - cofactors - before their catalytic
activity can be exerted. This entire active complex is referred to as the holoenzyme.

The cofactor may be:

1. A coenzyme - a non-protein organic substance which is dialyzable, thermostable and loosely

attached to the protein part.

2. A prosthetic group - an organic substance which is dialyzable and thermostable which is firmly
attached to the protein or apoenzyme portion.

3. A metal-ion-activator - these include K+, Fe++, Fe+++, Cu++, Co++, Zn++, Mn++, Mg++, Ca++ and Mo+++.

Specificity of Enzymes

1. Absolute specificity - the enzyme will catalyze only one reaction.

2. Group specificity - the enzyme will act only on molecules that have specific functional groups,
such as amino, phosphate and methyl groups.

3. Linkage specificity - the enzyme will act on a particular type of chemical bond regardless of the
rest of the molecular structure.

4. Stereochemical specificity - the enzyme will act on a particular steric or optical isomer.

22
Enzyme Kinetics

Enzymes are catalysts and increase the speed of a chemical reaction without themselves
undergoing any permanent chemical change. They are neither used up in the reaction nor do they
appear as reaction products.

The basic enzymatic reaction can be represented as follows

where E represents the enzyme catalyzing the reaction, S the substrate, the substance being
changed, and P the product of the reaction.

For most chemical reactions to proceed, some form of energy is needed and it is called "the energy
of activation". The magnitude of the activation energy determines how fast the reaction will proceed.
Enzymes are known to lower the activation energy for the reaction they are catalyzing.

The Enzyme Substrate Complex

The substrate and enzyme formed some intermediate substance which is known as the enzyme
substrate complex. The enzyme first unites in some way with the substrate and then returns to its
original form after the reaction is concluded.

Chemical Equilibrium

The study of a large number of chemical reactions reveals that most do not go to true completion.

This is likewise true of enzymatically-catalyzed reactions. This is due to the reversibility of most
reactions.

23
where K +1is the forward reaction rate constant and K-1 is the rate constant for the reverse
reaction.

Combining the two reactions gives:

Applying this general relationship to enzymatic reactions allows the equation:

Equilibrium, a steady state condition, is reached when the forward reaction rates equal the
backward.

Factors Affecting Enzyme Activity

Several factors affect the rate at which enzymatic reactions are temperature, pH, enzyme
concentration, substrate concentration, and the presence of any inhibitors or activators.

Enzyme Concentration

In order to study the effect of increasing the enzyme concentration upon the reaction rate, the
substrate must be present in an excess amount; i.e., the reaction must be independent of the substrate
concentration. Any change in the amount of product formed over a specified period of time will be
dependent upon the level of enzyme present. Graphically this can be represented as:

24
 These reactions are said to be "zero order" because the rates are independent of substrate
concentration, and are equal to some constant k. The formation of product proceeds at a rate which
is linear with time. The addition of more substrate does not serve to increase the rate. In zero order
kinetics, allowing the assay to run for double time results in double the amount of product.

The amount of enzyme present in a reaction is measured by the activity it catalyzes. The relationship
between activity and concentration is affected by many factors such as temperature, pH, etc. An
enzyme assay must be designed so that the observed activity is proportional to the amount of enzyme
present in order that the enzyme concentration is the only limiting factor. It is satisfied only when the
reaction is zero order.

In Figure, activity is directly proportional to concentration in the area AB, but not in BC. Enzyme
activity is generally greatest when substrate concentration is unlimiting.

When the concentration of the product of an enzymatic reaction is plotted against time, a
similar curve results,

25
 Between A and B, the curve represents a zero order reaction; that is, one in which the rate is
constant with time. As substrate is used up, the enzyme's active sites are no longer saturated, substrate
concentration becomes rate limiting, and the reaction becomes first order between B and
C. To measure enzyme activity ideally, the measurements must be made in that portion of the curve
where the reaction is zero order. A reaction is most likely to be zero order initially since substrate
concentration is highest. To be certain that a reaction is zero order, multiple measurements of product
(or substrate) concentration must be made.

Substrate Concentration

It has been shown experimentally that if the amount of the enzyme is kept constant and the
substrate concentration is then gradually increased, the reaction velocity will increase until it reaches
a maximum. After this point, increases in substrate concentration will not increase the velocity (delta
A/delta T).

It is theorized that when this maximum velocity had been reached, all of the available enzyme has
been converted to ES, the enzyme substrate complex. This point on the graph is designated Vmax.

Using this maximum velocity and equation

26
 Michaelis developed a set of mathematical expressions to calculate enzyme activity in terms of
reaction speed from measurable laboratory data.

The Michaelis constant Km is defined as the substrate concentration at 1/2 the maximum velocity.

Using this constant and the fact that Km can also be defined as:

K+1, K-1 and K+2 are the rate constants from equation. Michaelis developed the following
expression for the reaction velocity in terms of this constant and the substrate concentration.

Michaelis constants have been determined for many of the commonly used enzymes. The size of
Km tells us several things about a particular enzyme.

1. A small Km indicates that the enzyme requires only a small amount of substrate to become
saturated. Hence, the maximum velocity is reached at relatively low substrate concentrations.

2. A large Km indicates the need for high substrate concentrations to achieve maximum reaction
velocity.

3. The substrate with the lowest Km upon which the enzyme acts as a catalyst is frequently
assumed to be enzyme's natural substrate, though this is not true for all enzymes.

Displaying enzyme kinetic data on a Lineweaver- Burk plot

The best way to analyze enzyme kinetic data is to fit the data directly to the Michaelis-Menten
equation using nonlinear regression. Before nonlinear regression was available, investigators had to
transform curved data into straight lines, so they could analyze with linear regression.

One way to do this is with a Lineweaver-Burk plot. Take the inverse of the Michaelis-Menten
equation and simplify:

27
 Ignoring experimental error, a plot of 1/V vs. 1/S will be linear, with a Y-intercept of 1/Vmax and a
slope equal to Km/Vmax. The X-intercept equals ? 1/Km.

β-galactosidase

β-galactosidase is the enzyme hydrolyzes β−D-galactosidase. This enzyme facilitates growth on

carbon sources like lactose by cleaving it into a molecule of glucose and a molecule of galactose
which cells can catabolize and grow on. In the assay the substrate o-nitrophenyl-b-D-galactosidase
(ONPG) is used in place of lactose. When the β-galactosidase cleaves ONPG o-nitrophenol is released.
o-nitrophenol has a yellow color and it adsorbs light at 420nm light. Accumulation of yellow color is
monitored to measure the β-galactosidase activity.

28
Lab 12 Enzyme Kinetics 2
Inhibitor Effect
Enzyme inhibitors are molecular agents that interfere with catalysis, slowing or halting enzymatic
reactions. There are two broad classes of enzyme inhibitors: reversible and irreversible.

Reversible Inhibition

Enzyme can retain activity after removal of inhibitor molecule. One common type of reversible
inhibition is called competitive. A competitive inhibitor competes with the substrate for the active site
of an enzyme. While the inhibitor (I) occupies the active site it prevents binding of the substrate to the
enzyme. Many competitive inhibitors are compounds that resemble the substrate and combine with
the enzyme to form an EI complex, but without leading to catalysis.

Because the inhibitor binds reversibly to the enzyme, the competition can be biased to favor the
substrate simply by adding more substrate. When [S] far exceeds [I], the probability that an inhibitor
molecule will bind to the enzyme is minimized and the reaction exhibits a normal Vmax. However,
the [S] at which V0=1/2 Vmax, the apparent Km, increases in the presence of inhibitor by the factor .
This effect on apparent Km, combined with the absence of an effect on Vmax, is diagnostic of
competitive inhibition and is readily revealed in a double- reciprocal plot

Two other types of reversible inhibition, uncompetitive and mixed are in practice observed only
with enzymes having two or more substrates.

An uncompetitive inhibitor binds at a site distinct from the substrate active site and, unlike a
competitive inhibitor, binds only to the ES complex. An uncompetitive inhibitor lowers the measured
Vmax. Apparent Km also decreases, because the [S] required to reach one-half Vmax decreases by the
factor  .

A mixed inhibitor also binds at a site distinct from the substrate active site, but it binds to either E
or ES. A mixed inhibitor usually affects both Km and Vmax.

29
Irreversible Inhibition

The irreversible inhibitors are those that bind covalently with or destroy a functional group on an
enzyme that is essential for the enzyme’s activity, or those that form a particularly stable noncovalent
association.

pH Effect
Enzymes have an optimum pH (or pH range) at which their activity is maximal, at higher or lower
pH, activity decreases. Amino acid side chains in the active site may act as weak acids and bases with
critical functions that depend on their maintaining a certain state of ionization, and elsewhere in the
protein ionized side chains may play an essential role in the interactions that maintain protein
structure. Removing a proton from a His residue, for example, might eliminate an ionic interaction
essential for stabilizing the active conformation of the enzyme. A less common cause of pH sensitivity
is titration of a group on the substrate.

30
Temperature Effect
Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the
temperature is raised. A ten-degree Centigrade rise in temperature will increase the activity of most
enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce
changes of 10 to 20% in the results. The reaction rate increases with temperature to a maximum level,
then abruptly declines with further increase of temperature. Because most animal enzymes rapidly
become denatured at temperatures above 40oC, most enzyme determinations are carried out
somewhat below that temperature.

Goals of the experiment

Studying Factors Affecting Enzyme Activity

• pH

Set up 7 different reaction mixtures containing pH 4-5-6-7-8-9-10 Z buffers. Measure the activity.
Calculate the % activity compared with pH 7. Draw a graph between % activity and pH.

• Temperature

Set up 5 different reaction mixtures incubate the mixture at 4-RT-37-60-90oC. Calculate the %
activity compared with 30 oC. Draw a graph between % activity and temperature.

• Inhibitors

Set up 4 different reaction mixtures and add 0-7,5-10-15-20 mM Lactose to mixture before starting
the reaction. Calculate the % activity compared with 0 mM Lactose. Draw a graph between %
activity and Lactose concentration.

31
Lab 8 Carbohydrates
Carbohydrates are the most abundant organic molecules in nature. They have a wide range of
functions, including providing a significant fraction of the energy in the diet of most organisms, acting
as a storage form of energy in the body, and serving as cell membrane components that mediate some
forms of intercellular communication. Carbohydrates also serve as a structural component of many
organisms, including the cell walls of bacteria, the exoskeleton of many insects, and the fibrous
cellulose of plants. The empiric formula for many of the simpler hydrates is hence the name "hydrate
of carbon."

Monosaccharide (simple sugars) can be classified according to the number of carbon atoms they
contain.

Carbohydrates with an aldehyde as their most oxidized functional group are called aldoses,
whereas those with a keto group as their most oxidized functional group are called ketoses.

Monosaccharide can be linked by glycosidic bonds to create larger structures. Disaccharides

contain two monosaccharide units; oligosaccharides contain from three to about twelve
monosaccharide units, whereas polysaccharides contain more than twelve monosaccharide units, and
can be hundreds of sugar units in length.

32
 The form in which carbohydrates are distributed by the blood of vertebrates is glucose (“blood
sugar”). This is taken up by the cells and either broken down to obtain energy (glycolysis) or converted
into other metabolites. Several organs (particularly the liver and muscles) store glycogen as a
polymeric reserve carbohydrate. The glycogenmolecules are covalently bound to a protein,
glycogenin.
Polysaccharides that are formed from only one type of monosaccharide are called homoglycans,
while those formed from different sugar constituents are called heteroglycans.

33
 Glycogen, the reserve carbohydrate of higher animals, is stored in the liver andmusculature in
particular. The formation and breakdown of glycogen are subject to complex regulation by hormones
and other factors.

Reducing and Nonreducing Sugars: Because monosaccharides and most disaccharides are
hemiacetals and therefore contain a reactive carbonyl group, they are readily oxidized to diverse
products, a property often used in their analysis. Such carbohydrates, including glucose, maltose,
cellobiose, and lactose, are sometimes called reducing sugars. Historically, reducing sugars were
detected by their ability to reduce metal ions such as or to insoluble products. Carbohydrates that
are acetals, such as sucrose, are not readily oxidized because both anomeric carbon atoms are fixed
in a glycosidic linkage. These are classified as nonreducing sugars.

MATERIALS AND METHODS

Preparation of glycogen

75 g liver is transferred to 300 ml. of 30% (w/v) NaOH at 100 ºC. Heating was continued for 2 hr.
and the crude glycogen precipitated by adding twice the volume of 96% (v/v) ethanol. After separation,
the precipitate was dissolved in a small volume of water and the solution adjusted to pH 3 with dilute
HCI. The glycogen was re-precipitated by the addition of an equal volume of ethanol. This process of
precipitation was repeated once more, and the resulting substance washed with ethanol.

34
Determination of Glycogen

Quantitative Analysis (Phenol-Sulfuric acid Method)

0.5 ml 5% phenol + 0,5ml glucose + 2.5ml concentrate sulfuric acid

20 min incubation

Absorbance 470nm

Standards:

Stock glucose: 0.1M

Dilutions: 1/500, 1/200, 1/100, 1/50, 1/25

Sample Dilutions: 1/20, 1/40, 1/50, 1/100

Qualitative Analysis (Benedict’s Test)

2ml 0,02M glucose solution + 1,5ml Benedict’s solution

2ml of extracted glycogen + 1,5ml Benedict’s solution

Heat to 65 o C and observe the color changes

Blue (-)

Green (+)

Orange (++)

Red (+++)

Reddish Brown (++++)

35
 Lab 9 Lipids
Because the universal liquid milieu of living systems is water, you might expect that the chemical
components of cells would be polar substances that are readily soluble in water. This is generally the
case, but there exists a rather heterogeneous class of biological molecules that are soluble in nonpolar
solvents, such as benzene, ether, chloroform, or slightly more polar solvents, such as methanol or
acetone. This class is called lipids. It is perhaps most useful to consider the lipids by their functions. A
major function of the triglycerides (fats and oils) is storage and transport of metabolic energy.

Waxes, hydrocarbons, and fatty alcohols, the most nonpolar lipids, function primarily in forming
protective surfaces of plant and animal tissues, although waxes are used in place of triglycerides as
energy storage forms in some aquatic animals. The most important lipids are those that serve as
structural components of biological membranes (see Fig. VI). All of the members of this group
(phospholipids, glycolipids, sphingolipids, and sterols—the latter two are usually absent from bacterial
membranes) are amphipathic.

That is, they possess a nonpolar tail, often containing long-chain fatty acyl groups, and a polar head
group. Considerable attention will be paid in this section to these lipids and their role in membrane
structure and function. Finally, there is a large group of lipids of relatively low molecular weight
(steroids, terpenes, prostaglandins, and quinones) that have diverse and very importantfunctions as
hormones, vitamins, and photopigments,as well as other more specialized roles.

Structure and Classification of Lipids

Triacylglycerols and Related Simple Lipids

Triacylglycerols (triglycerides)—that is, glycerylesters of fatty acids (e.g., triolein and α-palmitoyl-
α’, β-diolein [Fig. I])—are by far the most abundant lipids in higher plants and animals. These lipids
accumulate in fatty depots and seeds as a storage form of food energy. Natural fats generally contain
a variety of fatty acids, and are best represented as “mixed” fats, as in the case of 1-palmitoyl-2,3-
diolein in Fig. I. Diglycerides and monoglycerides exist in various tissues, usually in small amounts.
Diglycerides play an important role in the biosynthesis of some of the more complex lipids and in signal
transduction in eukaryotic cells.

Figure 1 Structures of two

triglycerides.

36
 Sterols and Sterol Esters

The sterols are hydroxylated derivatives of the cyclopentanoperhydrophenanthrene (steroid)

nucleus (Fig. II). All sterols are capable of forming esters with fatty acids, but in general, the free sterols
are more abundant than their esters. The most abundant sterol is cholesterol, which is found in the
membranes of most animal cells (Fig.).

Figure 2 Structures of sterols.

When cholesterol occurs in the esterified forms, it usually is combined with common fatty acids,
such as palmitic, oleic, or linoleic acids. Plant sterols are occasionally esterified with acids, such as acetic
and cinnamic acids, or are incorporated into glycosides (saponins). Cholesterol is the precursor to many
important steroid hormones that regulate metabolism and reproduction. Well-known examples
include cortisol, aldosterone, progesterone, testosterone, and estradiol (see Fig. II), but there are many
others.

Glycerophosphatides

These lipids are mainly acyl derivatives of α-glycerophosphoric acid and often are called
“phospholipids.” The simplest glycerophosphatides are the phosphatidic acids, which contain α-
glycerophosphoric acid esterified with two fatty acids (Fig. III). Small quantities of phosphatidic acids
have been isolated from a wide variety of plant and animal tissues. It is doubtful that these compounds
exist in large amounts in tissues, because more complex glycerophosphatides are readily hydrolyzed
by enzymes that are widely distributed in such tissues, yielding phosphatidic acids. Phosphatidic acid
is a crucial intermediate in the biosynthesis of phospholipids.

Figure 3 A phosphatidic acid

37
 The major glycerophosphatides are esters of phosphatidic acid that have the general formula shown
in Figure IV, in which R represents a long-chain alkyl group and Rʹ an amino alcohol or inositol.
Additional phosphoryl groups are esterified to inositol in some phosphatidyl inositols. Phosphatidyl
inositol diphosphate (PIP2) and other phosphoinositols are important molecules in signal transduction.

Figure 4 Structures of various glycerophosphatides. The generic structure is shown at the top of
the figure, and the classes differ in the structure of the Rʹ group as shown.

Additional residues of glycerol are incorporated in the more complex structures of phosphatidyl
glycerol, phosphatidyl 3_-o-aminoacylglycerol (lipamino acid), and cardiolipin (see Fig. III-38). The
plasmalogens represent an unusual type of glycerophosphatide that has been isolated from animal and
plant tissues. Acid hydrolysis of these materials yields long-chain aldehydes and monoacylated _-
glycerophosphate. The long-chain aldehydes originate from vinyl ether structures. The vinyl ether
group appears usually (if not always) in the α-position. The Rʹ phosphomonoester group may be either
choline or ethanolamine, although ethanolamine usually predominates. In the structure of a
plasmalogen shown in Figure V, R is
an aliphatic chain and Rʹ is choline or ethanolamine.

Figure 5 Structure of plasmalogens.

38
 Figure 6
Estimated
Chemical
Compositions
of Some
Membranes

Thin-Layer Chromatography

Thin-layer chromatography (TLC) is a powerful, mainly analytic, technique for rapidly separating
lipids and other small molecules such as amino acids, nucleotides, vitamins, and drugs. The sample
being analyzed is applied to a plate made of glass, aluminum, or plastic, which is covered with a thin
layer of silica gel or other material. The plate is then placed in a chromatography chamber that contains
some solvent. Drawn by the capillary forces the solvent moves up the plate. The substances in the
sample move with the solvent. The speed at which they move is determined by their distribution
between the stationary phase (the hydrophilic silica), and the mobile phase (the hydrophobic solvent).
When the solvent reaches the top edge of the plate, the chromatography is stopped. After evaporation
of the solvent, the separated substances can be made visible using appropriate staining methods or
with physical processes (e. g., ultraviolet light). The movement of a substance in a given TLC system is
expressed as its Rf value. In this way, compounds that are not known can be identified by comparison
with reference substances.

A process in which the polarity of the stationary and mobile phases is reversed—i. e.,the stationary
phase is apolar and the solvent is polar—is known as “reversed-phase thinlayer chromatography” (RP-
TLC).

39
Isolation, Separation, and Analysis of Lipids

Solvent Extraction

The first step in the preparation of a lipid is extraction from the tissue. Since lipids are defined by
their solubility in organic solvents, whereas proteins, nucleic acids, carbohydrates and most of the low-
molecular weight metabolites of the cell are generally water soluble, the isolation of lipids is, in
principle, quite simple. Extraction of a biological sample with an organic solvent that is immiscible with
water, followed by separation of the phases should yield the lipid components in the organic phase.
The various classes of lipids can then be separated by the chromatographic techniques.

Lipid Extraction and TLC of Lipids

Solutions & Reagents

A chloroform/methanol 1:1 (v/v)

B chloroform/methanol 3:1 (v/v)

C chloroform/methanol/1,2 N HCl 10:10:1 (v/v/v)

Mobile solvents

1) chloroform/methanol/4.3Mammonia 90:65:20 (v/v/v)

2) n-propanol/4.3M ammonia 65:35 (v/v)
3) n-butanol/chloroform/glacial acetic acid/ddH2O 60:10:20:10 (v/v/v/v)
4) isopropylether/glacial acetic acid 96:4 (v/v)
5) petrol ether/diethylether/glacial acetic acid 90:10:1(v/v/v)
6) chloroform/methanol/ddH2O 65:25:4 (v/v/v)
7) n-butanol/glacial acetic acid/ddH2O 60:20:20 (v/v/v)
8) chloroform/methanol/acetone/glacial acetic acid/ddH2O 75:15:30:15:7.5 (v/v/v/v/v)

Extraction

Freeze the tissue, which should be extracted, with liquid nitrogen, and pulverize it in a mortar under
liquid nitrogen. Extract the tissue powder with 1 ml/g of Soln. A in a glass/Teflon homogenizator at RT.
Centrifuge at room temperature, decant the liquid, extract with 10 vol. of Soln. B, and centrifuge again
and homogenize with 2.5 vol. of Soln. C again.

Combine the liquid extracts and vaporize the solvent in a nitrogen atmosphere.

Apply the lipid-containing solution as a short line with a microliter syringe or a pipet. The start zone
should be as thin as possible. If larger volumes have to be applied, pipette several times ontothe
same position with intermediate drying. For drying the starting area as well as the plate after
chromatography uses a stream of nitrogen instead of air to avoid oxidation of unsaturated lipids.
Identify the unknown lipids by comparison with a set of known lipids (standards) of the same type,
chromatographed in the same run on the same plate.
A general chromatographic system for separation of lipids does not exist. Whereas sophisticated
systems for groups of lipids are described in literature, do not hesitate to try other solvent systems.

40
 Lipid detection
A versatile detection reagent for lipids is iodine vapor. The dry plate is placed into a chamber
containing one or two Petri dishes with crystals of solid iodine. Lipid-containing spots are colored
yellow to brownish after few minutes. The color disappears quickly in contact with air. An additional
general detection method is the oxidation by concentrated sulfuric acid. Spray the dry plate in a hood
with concentrated sulfuric acid and then heat it in a drying oven to 130 ◦C. Organic substances yield
dark brown spots. Lipids are visualized by spraying the plate with several other solutions from
literature.

41
Lab 10 Nucleic Acids
Ribonucleic acid (RNA) is a polymer of purine and pyrimidine ribonucleotides linked together by
3′,5′-phosphodiester bridges analogous to those in DNA. Although sharing many features with DNA,
RNA possesses several specific differences:

(1) In RNA, the sugar moiety to which the phosphates and purine and pyrimidine bases are attached is
ribose rather than the 2′-deoxyribose of DNA.

(2) The pyrimidine components of RNA differ from those of DNA. Although RNA contains the
ribonucleotides of adenine, guanine, and cytosine, it does not possess thymine except in the rare case
mentioned below. Instead of thymine, RNA contains the ribonucleotide of uracil.

(3) RNA exists as a single strand, whereas DNA exists as a

double-stranded helical molecule. However given the
proper complementary base sequence with opposite
polarity, the single strand of RNA is capable of folding
back on itself like a hairpin and thus acquiring double-
stranded characteristics.

(4) Since the RNA molecule is a single strand

complementary to only one of the two strands of a gene,
its guanine content does not necessarily equal its
cytosine content, nor does its adenine content
necessarily equal its uracil content.

(5) RNA can be hydrolyzed by alkali to 2′,3′ cyclic diesters

of the mononucleotides, compounds that can- not be
formed from alkali-treated DNA because of the absence
of a 2′-hydroxyl group. The alkali lability of RNA is useful
both diagnostically and analytically.

RNA SPECIES

A. Messenger RNA (mRNA)

This class is the most heterogeneous in size and stability. All members of the class function as
messengers conveying the information in a gene to the protein synthesizing machinery, where each
serves as a template on which a specific sequence of amino acids is polymerized to form a specific
protein molecule, the ultimate gene product.

42
 Messenger RNAs, particularly in eukaryotes, have some unique chemical characteristics. The 5′
terminal of mRNA is “capped” by a 7-methylguanosine triphosphate that is linked to an adjacent 2′- O-
methyl ribonucleoside at its 5′-hydroxyl through the three phosphates. The mRNA molecules
frequently contain internal 6-methyladenylates and other 2′-O-ribose methylated nucleotides. The cap
is involved in the recognition of mRNA by the translating machinery, and it probably helps stabilize the
mRNA by preventing the attack of 5′-exonucleases. The protein-synthesizing machinery begins
translating the mRNA into proteins beginning downstream of the 5′ or capped terminal. The other end
of most mRNA molecules, the 3′-hydroxyl terminal, has an attached polymer of adenylate residues 20–
250 nucleotides in length. The specific function of the poly(A) “tail” at the 3′-hydroxyl terminal of
mRNAs is not fully understood, but it seems that it maintains the intracellular stability of the specific
mRNA by preventing the attack of 3′-exonucleases. Some mRNAs, including those for some histones,
do not contain poly(A). The poly(A) tail, because it will form a base pair with oligodeoxythymidine
polymers attached to a solid substrate like cellulose, can be used to separate mRNA from other species
of RNA, including mRNA molecules that lack this tail.

In mammalian cells, including cells of

humans, the mRNA molecules present in the
cytoplasm are not the RNA products
immediately synthesized from the DNA
template but must be formed by processing
from a precursor molecule before entering the
cytoplasm. Thus, in mammalian nuclei, the
immediate products of gene transcription
constitute a fourth class of RNA molecules.
These nuclear RNA molecules are very
heterogeneous in size and are quite large. The
heterogeneous nuclear RNA (hnRNA)
molecules may have a molecular weight in
excess of 107, whereas the molecular weight
of mRNA molecules is generally less than 2 ×
106. hnRNA molecules are processed to
generate the mRNA molecules which then
enter the cytoplasm to serve as templates for
protein synthesis.

B. Transfer RNA (tRNA)

tRNA molecules vary in length from 74 to 95 nucleotides. They also are generated by nuclear
processing of a precursor molecule. The tRNA molecules serve as adapters for the translation of the
information in the sequence of nucleotides of the mRNA into specific amino acids. There are at least
20 species of tRNA molecules in every cell, at least one (and often several) corresponding to each of
the 20 amino acids required for protein synthesis. Although each specific tRNA differs from the others
in its sequence of nucleotides, the tRNA molecules as a class have many features in common.

43
 The primary structure, the nucleotide sequence of
all tRNA molecules allows extensive folding and
intrastrand complementarity to generate a secondary
structure that appears like a cloverleaf. All tRNA
molecules contain four main arms. The acceptor arm
terminates in the nucleotides CpCpAOH. These three
nucleotides are added posttranscriptionally. The tRNA-
appropriate amino acid is attached to the 3′-OH group
of the A moiety of the acceptor arm. The D, T C, and
extra arms help define a specific tRNA.Although tRNAs
are quite stable in prokaryotes, they are somewhat less
stable in eukaryotes. The opposite is true for mRNAs,
which are quite unstable in prokaryotes but generally
stable in eukaryoticorganisms.

C. Ribosomal RNA (rRNA)

A ribosome is a cytoplasmic nucleoprotein structure

that acts as the machinery for the synthesis of proteins
from the mRNA templates. On the
ribosomes, the mRNA and tRNA molecules interact to translate into specific protein molecule
information transcribed from the gene. In active protein synthesis, many ribosomes are associated
with an mRNA molecule in an assembly called the polysome. The components of the mammalian
ribosome have a molecular weight of about 4.2 × 106 and a sedimentation velocity of 80S (Svedberg
units).

The mammalian ribosome containstwo

major nucleoprotein subunits—a larger
one with a molecular weight of 2.8
× 106 (60S) and a smaller subunit with a
molecular weight of 1.4 × 106 (40S). The
60S subunit contains a 5S ribosomal RNA
(rRNA), a 5.8S rRNA, and a 28S rRNA;
there are also probably more than 50
specific polypeptides. The 40S subunit is
smaller and contains a single 18S rRNA and
approximately 30 distinct
polypeptide chains. All of the ribosomal RNA molecules except the 5S rRNA are processed from a single
45S precursor RNA molecule in the nucleolus. 5S rRNA is independently transcribed. The highly
methylated ribosomal RNA molecules are packaged in the nucleolus with the specific ribosomal
proteins. In the cytoplasm, the ribosomes remain quite stable and capable of many translation cycles.
The functions of the ribosomal RNA molecules in the ribosomal particle are not fully understood, but
they are necessary for ribosomal assembly and seem to play key roles in the binding of mRNA to
ribosomes and its translation. Recent studies suggest that an rRNA component performs the peptidyl
transferase activity and thus is an enzyme (a ribozyme).

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 D. Small stable RNA

A large number of discrete highly conserved, and small stable RNA species are found in eukaryotic
cells. The majorities of these molecules are complex with proteins to form ribonucleoproteins and are
distributed in the nucleus, in the cytoplasm, or in both. They range in size from 90 to 300 nucleotides
and are present in 100,000–1,000,000 copies per cell.

Small nuclear RNAs (snRNAs), a subset of these

RNAs, are significantly involved in mRNA processing
and gene regulation. Of the several snRNAs, U1, U2,
U4, U5, and U6 are involved in intron removal and the
processing of hnRNA into mRNA. The U7 snRNA may
be involved in production of the correct 3′ ends of
histone mRNA—which lacks a poly (A) tail. The U4and
U6 snRNAs may also be required for poly(A)
processing.

EXPERIMENTAL PROCEDURE:

Total RNA isolation with phenol extraction

a) Culture growth and sample collection

• Inoculate e. coli in to LB media.

• Let them to grow up to OD 0.4

• Take 2ml of cell culture and centrifuge at 13000g at 4 C and pellet the bacteria.

• Wash the pellet with PBS centrifuge at 13000g at 4 C and pellet the bacteria.

b) Lysis

• Resuspend pellet at 0.5 ml of lysis buffer (2% SDS and 4mM EDTA)

• Incubate the cells in a 100C water bath for 3 min to lyse the cells

• Add 15ul of 3M NaOAc to each tube and transfer on ice.

c) Phenol Extraction

• Add an equal volume (0.5ml) of water saturated phenol to each tube.

• Invert several times

• Transfer to 67C water bath for 6 min invert in every 40 sec.

• Immediately transfer to ice.

• Centrifuge for 10 min at 4 C at 15000g

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• Transfer the aqueause layer to a new tube. The aqueaus layer is the top layer and distinct from the
organic phase which is tinted yellow.

d) Phenol/Chloroform extraction

• Add 0.5 ml of water saturated phenol :chloroform:isolamyl alcohol (25:24:1) to each tube.

• Invert several times

• Centrifuge for 5 min at 4 C at 15000g

• Transfer the aqueause layer to a new tube

• Repeat phenol/chloroform extraction once

e) Ethanol Precipitation

• To each tube add 1/10 volume (50ul) 3M NaOAc, 1/10 volume (50ul) 1mM EDTA and 2-2.5 volume
of (1ml) cold 100% EtOH.

• Invert several times then incubate at -80 C for 20 min.

• Centrifuge for 25 min at 4 C at 15000g

• Decant EtOH from pellets and discard

• Wash pellet in 1ml cold 80% EtOH.

• Centrifuge for 5 min at 4 C at 15000g

• Decant EtOH from pellets and discard

• Repeat wash with cold 80% EtOH twice for total 3 washes.

• Dry the pellet in a 37 C heat block after the final wash is removed for 15 min.

• Resuspend cell pellet at 50ul EB buffer

f) Absorbance reading

• Measure the RNA concentration in nanodrop spectrophotometer.

g) Run the RNA in agarose gel

• Prepare 1% agarose gel containing ethidium bromide.

References

Harper’s Illustrated Biochemistry, twenty-sixth edition

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