Cell/Tissue-Biomaterial Interaction
Practical course
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CONTENTS
General issues
GENERAL Laboratory safety
Lab Safety Contract
Lab 1. Protein visualization
Lab 2. Protein visualization (cont)
Lab 3. Protein purification
Lab 4. Protein Characterization
Lab 5. Protein adsorption
GENERAL ISSUES
Grading
Attendance (individually): 20%
Lab report (in group): 60%
Lab activity (individually): 20%
Code of conduct
Students must understand the Laboratory safety before starting this course
Absence without permission is not allowed
Group report must be submitted 1week after class
GENERAL Laboratory safety
The laboratory should provide students and instructors alike with an environment conducive to
accomplishing specific scientific tasks. It is imperative that everyone involved in the laboratory
recognize the importance of safety in the laboratory. In addition, everyone using the laboratory must be
aware of potential safety hazards such as faulty electrical outlets, frayed wires, broken glassware and
slides, chemical spills, and potentially dangerous organisms. Students must follow proper safety practices
in the laboratory and immediately report any safety hazards or accidents to the instructor.
Risk is identified as a substance or biological agent that might be harmful under specific circumstances
while hazard is the ability of a substance or biological agent to cause harm. In any case, a person working
with chemicals, bio-chemicals or other related agents should follow strictly certain principles applied in
each laboratory, and all practical works must be carried out with safety in mind to minimize the risk of
harm to yourself and to others. The followings are the basic rules for laboratory work.
1. Make sure that you know what to do in case of fire, including exit routes, how to raise the alarm,
and where to gather after leaving the building. Remember that the most important consideration
at all time is human safety.
2. All accidents, no matter how minor, must immediately be reported to the teacher.
3. Follow tutor’s instructions and the laboratory principles while doing lab work.
4. Use only materials and equipment that the teacher has authorized.
5. Follow the teacher’s written and verbal instructions closely and carefully.
6. Chemical goggles should be used when working in the laboratory and using chemicals.
7. Prepare for each laboratory activity by reading all instructions beforehand. Follow all directions
carefully and intelligently. Make note of any deviations announced by your instructor.
8. Use eye wash for any chemical contact with your body. It is located at sink in the wet lab next
door. There is also a full body shower just outside the door of this lab.
9. Wear lab-coat and closed footwear at all time.
10. Do not smoke, eat or drink in laboratory because of the risks of contamination by inhalation or
ingestion.
11. Know the location of the emergency exit, first aid, and firefighting-equipment.
12. Know the proper fire drill procedure and where to get help fast.
13. Never taste, smell, or touch chemicals unless specifically instructed to do so.
14. Take great care in noting odors or fumes. Use a wafting motion of the hand. Never put a bottle to
your noise and breathe deeply.
15. Do not mouth-pipette any liquid.
16. Take care when handling glassware.
17. Broken glass should be removed from work areas and the floor as soon as possible. Never handle
broken glass with your bare hands. Broken glass should be placed (using proper techniques) in
the labeled, sharps box.
18. All solid waste should be placed in separate waste containers, jars or other designated receptacle.
Do not discard any solids in the laboratory sinks.
19. Do not bring any substance into contact with a flame unless specifically instructed to do so.
20. Upon first entering the laboratory, students are not permitted to touch laboratory equipment until
directed to do so.
21. Always twist and never push glass tubing into stopper holes. Lubricate stopper holes and glass
tubing with glycerin to insert easily. Always use glass tubing with fire polished ends.
22. Never pour water into acid. Correctly dilute acid by pouring it into water.
23. Never allow the open end of a heated test tube to be pointed toward anyone.
24. Horseplay, running, pushing, shoving, and practical jokes will not be tolerated.
25. Conduct yourself in a responsible manner at all times in a laboratory situation.
26. Know the warning symbols for specific chemical hazards (see below).
27. Use fume cupboard for hazardous chemicals.
28. Work in a logical, tidy manner and minimize risks by planning in advance.
29. Clean up working bench and experimental tools at the end of each lab session.
Lab Safety Contract
Agreement
I, ___________________________________________, have had the safety training for the
biomedical engineering laboratory and agree to follow these safety regulations while in class.
___________________ ____________
(Student’s Signature) (Date)
Lab 1. Protein visualization
I. Introduction
PyMOL is an excellent open-source molecular visualization tool for structural biology and
bioinformatics. It is commonly used to examine protein structures, produce high-quality molecular
visualizations, and investigate interactions between biological macromolecules such as proteins, nucleic
acids, and ligands. PyMOL supports a wide range of visualization tools, including ray tracing,
animations, and movie generation, all of which are required for molecular modeling and publication-
quality images.
II. Installing PyMOL
1. System Requirements
- Windows: PyMOL runs on Windows 10/11 (64-bit versions).
- macOS: Compatible with macOS 10.14 and later.
2. Installing PyMOL (Windows and macOS)
Download PyMOL:
- Go to the official PyMOL website: Schrödinger's PyMOL (https://pymol.org/2/)
- Choose your platform (Windows or macOS) and download the appropriate installer.
Installation Process (Windows):
- Run the downloaded `.exe` file.
- Follow the installation prompts (click `Next` to proceed with default settings).
- Complete the installation by clicking `Finish`.
Installation Process (macOS):
- Download the `.dmg` file for macOS.
- Open the `.dmg` file and drag the PyMOL icon into the `Applications` folder.
3. Launching PyMOL
- Open PyMOL from the desktop shortcut or Applications folder.
III. Exercises
Exercise 1: Downloading a PDB File
1. Navigate to the Protein Data Bank:
- Open a web browser and go to RCSB PDB (http://www.rcsb.org).
2. Search for a Structure:
- In the search box, enter the PDB ID code `1FJS` and click 'Search'.
- Download the Biological Assembly file from the download options.
Exercise 2: Opening PyMOL and Loading a Structure
1. Launching PyMOL:
- Open PyMOL by double-clicking the application icon on your desktop.
2. Loading the PDB File:
- Type the following commands in the PyMOL command line:
```
cd Desktop
load 1FJS.pdb
```
- Alternatively, you can use the menu: `File > Open`, navigate to the downloaded `1FJS.pdb` file,
and open it.
Exercise 3: PyMOL Interface and Basic Controls
- Rotation: Left-click and drag to rotate the molecule.
- Zoom: Right-click and drag to zoom in or out.
- Translation: Middle-click and drag to move the molecule.
Exercise 4: Changing Molecular Representations
1. Cartoon Representation:
- Select the molecule in the “Names Panel” and choose `Show > Cartoon`.
2. Changing Colors:
- To color by secondary structure (helix, sheet, loop), click the molecule name and navigate to
`Color > by ss > Helix Sheet Loop`.
3. Highlighting Ligands:
- If a ligand is present, show it as spheres by selecting `Show > Spheres`.
Exercise 5: Saving Images
1. Creating a Publication-Quality Image:
- After arranging the molecule, type the following command in the command line to improve
image quality:
```
ray
```
2. Save the Image:
- Use `File > Save Image`, and save the image as `PNG` with your desired resolution.
Exercise 6: Introduction to Scripting
1. Creating a Simple Script:
- Open a text editor and type the following commands:
```
load 1FJS.pdb
show cartoon
color red, ss h
color yellow, ss s
```
2. Running the Script:
- Save the file as `myscript.pml` on your desktop. In PyMOL, type:
```
@myscript.pml
```
Exercise 7: Creating a Simple Animation
1. Set up the rotation:
```
mset 1 x36
util.mroll(1,36,1)
```
2. Play the Animation:
- Use the Play button in the PyMOL GUI to watch the molecule rotate.
Lab 2. Protein visualization (cont)
In this Exercise we will use the visualization program PyMOL and also have a brief look at the PDB, the
home of all experimental protein structures.
1. Open the internet browser and go to http://www.uniprot.org/uniprot/O15527. Here you can find
information on the protein “8-oxoguanine DNA glycosylase”, human OGG1. In the isoform -
OGG1 (Isoform 1A) there are 345 residues. The sequence is given by:
In the “Structure” section on the page you will find links to 3D structure databases (in the “Links”
column). What is the PDB identifier for the structure with the best resolution? What is the
resolution for structure 1EBM? Are any of the structures for the full-length protein? Are there any
visible hydrogen atoms in any of the structures? Why not?
2. Open a browser window and go to the PDB at http://www.rcsb.org.
Here you can search for protein structures determined by either X-ray crystallography or NMR. There
are various ways to search the database, the most common being by protein name, acronym, organism,
protein class or author name.
3. Type in 1EBM in the search field and press enter to search for this structure. You get only one
hit, so you are sent directly to the “Structure Summary” for this structure. Which
macromolecules does this structure contain? How many chains are there? What is chain A? Is it
mutated? (Now, in the fall of 2018, the database claims that the protein in not mutated. This is a
change since the spring, and the last 15 years, and is wrong! It is mutated, Lys249 (K on blue
background on previous page) to Gln (see sequence alignment below). This is clearly stated in the
original article. This is good example of an error in partially automatically generated databases).
Do you have any suggestions for why the researchers behind this study have mutated a single
residue in the middle of the sequence? If not, you will have the chance to guess later in this
exercise. Is it a full- length protein? What is missing? Do you have any suggestions for why it is
not full- length? (Hint: Structural disordered regions are hard to crystallize)
4. Click on “Download Files” (up at the right hand side) and download the “PDB Format” file.
Save it, for example on the Desktop, as 1EBM.pdb. Take a look at the pdb-file, for example in
WordPad or nano. It shows among other things the experimentally determined positions (atomic
coordinates) of all the atoms in the model (including many water molecules). Who are the
authors of this work? Is this an X-ray crystallography or NMR study? What is the resolution of
the structure? What do you find in the fields “REMARK 200”? Did they use a synchrotron for
this work? What do you find in the fields “REMARK 465” and “REMARK 470”? Can you
explain the missing residues/atoms? What do you find on the “SEQRES” lines?
5. (Open structure file in PYMOL, show cartoon) What is the green ball you see in the structure?
You might need to show “sequence” and click on the green ball to get an idea
6. Play around with the protein structure using the mouse buttons (left = rotate, middle press =
move, middle scroll = clipping, middle click = centring and right = zoom). If you manage to
“get lost in protein space” you can always centre the molecule again by “middle-clicking” with
the mouse at any given amino acid in the sequence display on the top. You can also “right-click”
on the background and choose “zoom (vis)”.
7. The isolated red crosses/stars are single water molecules that are fixed in the crystal near the
protein surface. We don’t need them right now, so press “H” for hide and go down to the waters
item on the pop-up list (this is just next to “1ebm” in the upper right corner of the viewer).
8. Actually, let’s turn off the whole protein. Select “H” everything.
9. Select “S” for show, then select cartoon. Now you only see the trace of the main chain of the
protein. You can also see the DNA duplex bound to OGG1. Select a nice colour by pressing “C”
and then pick one from the list according to your liking. Try one of the “spectrum” “rainbow”
colouring schemes as well.
10. Use “H” everything again and then “S” and choose (hiding everything again when you want
to)
i. “lines”. This is “wireframes”. Colour “gray 60” and then “by element”, first option.
Now you see carbon atoms as gray, oxygens as red and nitrogens as blue.
ii. “sticks”.
iii. “ribbon”. This is often called a “Cα-trace” and is a line connecting all the Cα atoms.
iv. “spheres”. This is CPK space-filling spheres. Choose a nice colouring scheme.
v. “surface”. You get the full surface of the protein complex. Choose a nice colouring
scheme.
11. Hide everything and show as cartoon. Colour according to secondary structure “by ss”. How
many β-sheets do you find? How many β-strands does each sheet contain? Are there more α-
helices or β-sheets in OGG1? Scroll the sequence in the PyMOL Viewer window (at the top) and
locate the residue Asp174. Click on this residue in the sequence (i.e. click on the “D”). The “D” is
now highlighted, it is “selected”. Numbering in the sequence is a bit awkward... Residue 171 is
Ile and residue 176 is Val. You will also find little pink squares on the structure corresponding to
this selection. Finally, you find the selection as “(sele)” in a separate row on the right hand side of
the Viewer window. For this selection choose “A” “rename selection” and type the name
“Asp174” before you press enter. If you click on “(Asp174)” the little pink squares disappear,
but you can still use this selection later.
12. Show the whole structure as “cartoon” and Asp174 as “sticks”. Zoom in on Asp174 and choose a
colouring that makes it easy to see. What kind of secondary structure element is Asp174 located
in?
13. Hide “everything” (for 1ebm) and then show as “sticks”. Colour “gray 60” and then “by element”
(first option in the list). You see some residues sticking out into the solvent from the surface of the
protein. Would you describe the side chains of these residues as hydrophilic or hydrophobic?
14. Choose one of these residues by clicking on it. You have made a new selection “(sele)”. For
“(sele)”, do “L” “residues” to get this residue labelled. Which residue is it? Which amino acid is
it?
15. Can you find any areas within the structure that are rich in hydrophobic residues? What forces are
stabilizing the structure in these regions?
16. The segment “NIARITGMVERLCQAFG” is a part of OGG1, residues 151-167. Go to
http://emboss.bioinformatics.nl/cgi-bin/emboss/pepwheel and generate a helical wheel plot
with this sequence (Use default settings). Does the helical wheel plot suggest/hint that this is an
α-helix or a β-sheet in this enzyme? Why? Can you find the sequence in the structure? Is it an
α-helix or a β-sheet? Which part of the segment is facing the solvent? What do we call
something that is hydrophobic on one side and hydrophilic on the other?
The helical wheel plot gives,
17. Select the residues 151-167. Display only this alpha helix and hide everything else. Show as
“sticks” and colour “by element”. Approximately how many turns are there in this helix? Which
atoms are involved in H-bonds stabilising the α-helix? There are two residues involved in each of
these H-bonds. How many residues is it between them in the sequence? Can you locate the bonds
which define the rotations phi and psi? Where are the Cα atoms? It may be easier to answer the
questions if you first do “Hide” “Side chain” to see only the main chain atoms. You can let
PyMOL make an attempt on localizing H-bonds by doing “Actions” “find” “polar contacts”
“within selection”. As you see, there are too many H-bonds. You may fix this by typing (in the
Viewer window at the command line/lower left corner after “PyMOL>” the following: “set
h_bond_max_angle, 30”. If you repeat the “find polar contacts” procedure you should now get a
slightly better result
18. Show the whole protein again. Far down on the right-hand side you see “selecting
residues”. That means that if you click on the structure, you can choose single residues.
Click on the word “selecting” until you get “selecting chains”. Click on a part of the
protein in the main window and confirm by looking at the sequence bar that you have
chosen the full “chain A” and nothing more.
19. Create a new object by typing “create OGG1, sele”. Click on “1ebm” to make this
object inactive. You now only see your new object “OGG1” in the main window.
Locate the N-terminus and the C-terminus. Are there any gaps in the protein
backbone? Which residues are missing? Why are they missing?
20. For the object OGG1, select “H” for hide, and then select everything. The molecule
disappears.
21. For the same object, select “S” for show, and then select surface. A nice protein
surface is calculated and displayed.
22. Active sites are often located in grooves or holes in the surface. Where do you think
the active site is located?
23. This protein is a DNA glycosylase that scans DNA and recognises and removes 8-
oxoguanine (8-oxoG), which is guanine (G) with an oxygen atom bound at position 8
on the DNA base. The protein belongs to the helix-hairpin-helix superfamily of DNA
glycosylases. Make 1EBM active again and hide everything in this object. Create a new
selection that contains the DNA chains. For this selection choose “A” “rename
selection” and type the new name “DNA” before you press enter. Use “S” for show,
and then select “sticks” for “DNA”.
24. DNA containing 8-oxoG is the substrate for this enzyme. Can you now locate the
active site?
Lab 3. Protein purification
I. Introduction
Protein purification is a sequence of steps used to extract one or more proteins from a
complex mixture, typically cells, tissues, or complete organisms. Protein purification is
critical for determining the function, structure, and relationships of the protein of interest.
Fibronectin is a large glycoprotein found in the extracellular matrix and blood plasma. It is
crucial for tissue repair, wound healing, and cell adhesion. By taking advantage of the
differences in protein solubility in salt solutions, it can be separated based on how soluble it
is in ammonium sulfate.
Ammonium sulfate precipitation is a common method to purify proteins. The principle is
based on salting out, where proteins become less soluble in water as salt concentration
increases, eventually precipitating out of solution. Different proteins have different solubility
thresholds, allowing selective precipitation.
II. Materials and Reagents
1. Human plasma (fresh or frozen, 5–10 mL)
2. Ammonium sulfate (solid)
3. Tris-HCl buffer (0.05 M, pH 7.5)
4. Ice, pipettes, and tips
III. Equipment
1. Beakers (100 mL, 250 mL)
2. Centrifuge and tubes
3. Dialysis tubing
IV. Protocol
Part A: Comparison of Ammonium Sulfate Concentrations
1. Plasma Clarification:
- Centrifuge human plasma at 3,000 x g for 10 minutes at 4°C to remove debris.
Collect the supernatant.
2. Divide the Plasma:
- Divide the clarified plasma into three equal parts (e.g., 2 mL each) into three
separate tubes.
3. Prepare Ammonium Sulfate Solutions:
- Tube 1: Add solid ammonium sulfate to achieve 40% saturation.
- Tube 2: Add ammonium sulfate to reach 60% saturation.
- Tube 3: Add ammonium sulfate to reach 80% saturation.
4. Precipitation:
- Stir each solution on ice for 30 minutes.
- Centrifuge all tubes at 10,000 x g for 20 minutes at 4°C.
- Collect the supernatants and pellets for analysis.
Part B: Fibronectin Purification at 40% Saturation
1. Precipitation at 40% Saturation:
- Add ammonium sulfate to the remaining plasma to reach 40% saturation.
- Stir for 30 minutes on ice.
- Centrifuge at 10,000 x g for 20 minutes at 4°C to pellet fibronectin.
2. Resuspension:
- Resuspend the fibronectin pellet in Tris-HCl buffer (pH 7.5).
3. Dialysis:
- Dialyze the resuspended pellet overnight against Tris-HCl buffer to remove
residual ammonium sulfate.
V.
Lab 4. Protein characterization
I. Introduction
Protein characterization is the process of identifying and defining the structure,
characteristics, and biological function of proteins. Protein characterization analysis includes
determining protein purity, molecular weight, structure, post-translational alterations, and
interactions, among other factors.
SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) is a discontinuous
electrophoretic technology created by Ulrich K. Laemmli that is widely used to separate
proteins with molecular masses ranging from 5 to 250 kDa. The combination of sodium
dodecyl sulfate (SDS) and polyacrylamide gel reduces the influence of structure and charge,
and proteins are classified based on their size.
II. Materials and Reagents
1. Tris-HCl (1.5M, pH 8.8)
2. Tris-HCl (1M, pH 6.8)
3. Acrylamide 30%
4. SDS 10%
5. APS 10%
6. TEMED
7. Isopropanol
8. Running buffer (1X)
9. 6X Laemmli buffer
10. Staining buffer
11. Distaining buffer
III. Equipment
1. SE260 Vertical Protein Electrophoresis Unit
2. Multiple gel caster
3. Biosystem AzureC300 Imaging System
IV. Protocol
1. Thaw the samples at 80oC in 10’
2. Assemble slab gel unit and check leakage
3. Prepare resolving gel by adding in order:
- Distilled water: 4.15ml
- Acrylamide 30%: 2.15ml
- Tris-HCl (1.5M, pH 8.8): 2.5ml
- SDS 10%: 100µl
- APS 10%: 100µl
- TEMED: 10µl
4. Immediately put resolving gel in the slab gel unit, then put isopropanol and wait 15’
5. Discard the isopropanol
6. Prepare stacking gel by adding in order:
- Distilled water: 2.1ml
- Acrylamide 30%: 0.5ml
- Tris-HCl (1.5M, pH 8.8): 0.38ml
- SDS 10%: 30µl
- APS 10%: 30µl
- TEMED: 3µl
7. Immediately put stacking gel in the slab gel unit, apply the comb and wait 15’
8. Prepare 8 running samples: 8ul sample + 2ul 6X Laemmli buffer (ratio 4:1) for each
sample
9. Remove the comb and put the slab gel unit into running chamber, then fill the
chamber with running buffer
10. Load 8µl sample ladder in first dwell, then load 10µl running samples in the
following dwells
11. Run the chamber at 100V – 20mA – 2.5h
12. Collect the gel and soak into staining buffer for 10’, then soak into distaining buffer
13. Take image of the result using the image system
Lab 5. Protein adsorption
I. Introduction
Protein adsorption is the first process that occurs after implantation of a biomaterial in the
human body. This process changes the properties of the surface and can induce structural
alterations on the adsorbed/desorbed proteins. Cell-biomaterial interactions are mediated by
the type, amount and conformation of the adsorbed proteins that can interact with specific
membrane receptors expressed by the cells. In this lab session, you will conduct experiment
to determine fibronectin adsorption on material surface using solute-depletion technique and
Bradford assay for determining protein concentration.
The Bradford assay is a protein determination method that involves the binding of Coomassie
Brilliant Blue G-250 dye to proteins (Bradford 1976). The dye exists in three forms: cationic
(red), neutral (green), and anionic (blue) (Compton and Jones 1985). Under acidic conditions,
the dye is predominantly in the doubly protonated red cationic form (A max = 470 nm).
However, when the dye binds to protein, it is converted to a stable unprotonated blue form
(Amax = 595 nm) (Reisner et al. 1975, Fazekes de St. Groth et al. 1963, Sedmack and
Grossberg 1977). It is this blue protein-dye form that is detected at 595 nm in the assay using
a spectrophotometer or microplate reader
II. Materials and Reagents
1. Plasma fibronectin
2. Bovine Serum Albumin (Sigma-Alrich) 2mg/ml
3. Bradford assay kit (Thermo Fisher Scientific, Catalog number 23236)
4. 4-well chambered slides (Ibidi)
5. Phosphate buffer saline (PBS) pH 7.4
III. Equipment
1. Microplate reader (Varioskan, Thermo Fisher Scientific)
IV. Protocol
1. Fibronectin adsorption assay
- Pipet 200µl of Fibronectin into wells of chambered slides and incubate for 15, 30,
45 min at 37˚C
- After incubation time, remove and retain supernatant for protein determination by
Bradford assay
2. Preparation of BSA standard
Use Table 1 as a guide to prepare a set of protein standards. Dilute the contents of one
Albumin Standard (BSA) ampule into several clean vials, preferably in the same
diluent as the sample(s). Each 1mL ampule of Albumin Standard is sufficient to
prepare a set of diluted standards for either working range suggested in Table 1. There
will be sufficient volume for three replications of each diluted standard.
Make your calculation
Microplate protocol
1. Pipette 150µL of each standard and unknown samples into the appropriate microplate
wells.
2. Add 150µL of the Coomassie Plus Reagent to each well and mix with plate shaker for 30
seconds.
3. Remove plate from shaker. For the most consistent results, incubate plate for 10 minutes at
room temperature (RT).
4. Measure the absorbance at or near 595nm on a plate reader.
5. Subtract the average 595nm measurement for the Blank replicates from the 595nm
measurements of all other individual standard and unknown sample replicates.
6. Prepare a standard curve by plotting the average Blank-corrected 595nm measurement for
each BSA standard vs. its concentration (µg/ml). Using the standard curve, determine the
protein concentration estimate for each unknown sample. Note: If using curve-fitting
algorithms associated with a microplate reader, a four-parameter (quadratic) or best-fit curve
will provide more accurate results than a purely linear fit. If plotting results by hand, a point-
to-point curve is preferable to a linear fit to the standard points.
