Loading Control

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Handbook

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Common Loading Control Proteins for Western Blot

kDa Whole Cell Cytoskeletal Mitochondrial Nuclear Serum

120 Viniculin

110

75 Transferrin

65 Lamin B1

60 HSP60

Alpha-tubulin Alpha-tubulin HDAC1

50 Beta-tubulin Beta-tubulin

Actin Actin

40 Beta-actin Beta-actin

35 GAPDH

VDCA1/porin PCNA

25

20 Cyclophilin B Cofilin

15 COX IV Histone H3
TABLE OF CONTENTS

1 Introduction to Loading Controls

2 How to Normalize Your Western Blot Data

3-5 What to Consider When Using a Loading Control Antibody

Detection Range
Concentration
Stripping and Reprobing

6 Summary: Attributes of an Effective Loading Control Protein

7-8 Protocols for Probing Loading Control Expression

Method 1: Stripping and Reprobing

Method 2: Cutting the Membrane
 Introduction to
Loading Controls

A Loading Control is Important for Data Normalization

Western blotting is a common laboratory technique used to detect and quantify target protein expression in a complex cell or
tissue lysate. This technique provides a semi-quantitative method for the comparison of relative target protein levels across
multiple samples. To generate accurate expression data by Western blot analysis, a loading control protein is required. Loading
controls provide a means to ensure equal protein loading across wells, as well as a reference point for data normalization.

Prior to western blot analysis, the total amount of sample protein is measured using a biochemical assay (e.g. Bradford, BCA,
etc.). Total protein measurements are then used to load equivalent amounts of protein across all wells of the gel. Equal protein
loading is required to make accurate comparisons of target protein expression data. Despite experimental measures to prevent
unequal loading, subsequent protocol steps, including uneven transfer from gel to membrane, can affect total sample protein. To
verify equal loading of the gel and even transfer from gel to membrane, a second control is often required.

A LOADING CONTROL PROTEIN ALLOWS

COMPARISON OF TARGET PROTEIN
LEVELS ACROSS MULTIPLE SAMPLES

A loading control protein confirms equal sample loading and gel-to-membrane transfer. Following target protein detection, a
loading control protein is detected and its expression is used to mathematically compensate for sample-to-sample
variation. Normalized expression is then compared across samples to confirm that changes in protein of interest expression
represent real differences and are not the result of differences in total protein abundance from sample-to-sample.

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How to Normalize Your
Western Blot Data

Normalizing Western Blot Data

Data normalization is required to accurately compare target protein expression across multiple samples in Western blot analysis.
IMAGES FOR NORMALIZATION SECTION
To normalize target protein expression, the band intensity of each sample is determined by densitometry. Next, the intensity of
the target protein is divided by the intensity of the loading control protein. This calculation adjusts the expression of the protein
of interest to a common scale and reduces the impact of sample-to-sample variation. Relative target protein expression can then
IMAGES
IMAGES FOR
FOR NORMALIZATION
NORMALIZATION SECTION
SECTION
be compared across all lanes to assess changes in target protein expression across samples.

Step 1 Step 2 Step 3

Determine the intensity of each Normalize the target protein levels Compare relative target protein
band using densitometry software NORMALIZED
to DENSITY
the loading control =
protein FOLD
levelsDIFFERENCE =
across samples

= *=
Loading Control Normalized Density
NORMALIZED
NORMALIZED DENSITYDENSITY FOLD DIFFERENCE
FOLD DIFFERENCE = =

( )
(lane 1) (each lane)
Target
Protein
x
Loading
Loading Control *
ControlControl
Loading * Normalized
Normalized
Normalized Density
Density *
Density

( ( )x )x
(lanelane)
(each 1)(lane 1) (each lane)
(each
(lane 1)lane)
TargetTarget
Target Protein
Protein Loading ControlControl
Loading Normalized *
Density Density
Normalized *
(each lane)
(each lane) (lane 1) (lane 1)
36 20 2 52 36
Target
Target = 1.0 *
LANE 1 = 36 X = 36 * LANE 1 =
Control 36 3620 202 2 52
52 36 36
52 36
= 136= X 36 X
LANE 1LANE = 36 =* 36 * LANE 1LANE
= 1 = = 1.0 *= 1.0 *
52
Control
Control 46 47 5252 52 36 22.636
LANE 2 = 20 X = 22.6 LANE 2 = = 0.63
52 5246 46
47 47 46
52 52 22.6 3622.6
= 220 = X 20 X
LANE 2LANE = 22.6= LANE 2LANE
= 2 = = 0.63 = 0.63
* The first lane is loaded 22.6
46
52 36 2.236
with the control sample. Data in lane 1 46
2 X = 2.2 LANE 3 = 2.2 = 0.06
should be used to normalize expression LANE 3 = 52
data in lanes 2 and 3. 47 52
= 2.2 LANE 3 = 362.2
= 0.06
= 2 X 2
LANE 3LANE X47 = 2.2 LANE 3 36= = 0.06
3 =
47 36

Normalizing Western Blot Data: After band intensity is determined by densitometry software, the loading control protein
is used to normalize target protein expression. To normalize expression, multiply the density of the target protein in each
lane by the ratio of loading control density from the control sample (lane 1) to the loading control density of other lanes.
The fold change can then be calculated by dividing the normalized expression from each lane by the normalized
expression of the control sample in lane 1.

Note: Expression data determined by Western blot analysis is semi-quantitative. Protein abundance is expressed relative to the
loading control protein and does not indicate the absolute amount of target protein in your sample.

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 What to Consider When Using
a Loading Control Antibody

Detection Range and Gel Overloading

An ideal loading control demonstrates a linear response over a wide range of sample concentrations. This means the intensity of
IMAGES
the loading control protein should increase in equal proportion with sample FOR DETECTION
concentration. For accurate RANGE SECTION
normalization of
Western blot data, both the protein of interest and loading control protein should fall within the linear range of detection.
Because many target proteins are expressed at low abundance relative to loading control proteins, large amounts of total protein
are often loaded on the gel to accurately detect the target protein. However, loading high amounts of total protein often places
the loading control outside of the linear range of detection. When this happens, the expression of the loading control protein is
no longer proportional to the amount of total protein present in the sample. Deceivingly, overloaded gels often give the
Protein
Load:
appearance of uniform loading control band densities across all lanes. However, this uniformity is not due to equal protein
IMAGES
loading, but rather samples FOR
exceeding DETECTION
the transfer capacity ofRANGE SECTION
the membrane or falling outside the linear range of detection.
Amt of
Measured Linear
Sample
Density Response
Loaded
The optimal amount of total protein to load on the gel should be determined to ensure the accurate quantification of both the
0.5 ng 10811 10811
target and loading control proteins. To determine the linear range of your samples, create a1.0standard
ng
curve
26158
using21622
serial dilutions
of your sample. The standard curve can then be used to identify the saturation point of the2.0
assay
ng and 48585
the total amount
43244 of
starting protein required. 4.0 ng 71737 86488
Protein
GES FOR DETECTION RANGE SECTION
8.0 ng 79795 172976
Load:
16.0 ng 75830 345952

Amt of
Measured Linear
Sample
Density Response
Loaded
0.5 ng 10811 10811 Linear
Density

Response
1.0 ng 26158 21622
Measured
2.0 ng 48585 43244
Density
4.0 ng 71737 86488
Protein
8.0 ng 79795 172976
Load:
16.0 ng 75830 345952 Protein Load

Amt of
Measured Linear
Sample
Density Response
Loaded
Linear
Determining 10811 Comparison
0.5 ng Linear Range: 10811 of the densitometry readings of each sample to the linear response indicates
Density

Response

the signal becomes saturated

1.0 ng 26158 above 4.0 ng. Above 4.0 ng, the band intensity
21622 is no longer proportional to total protein
Measured
Density
abundance:
2.0 ng the signal falls outside43244
48585 the linear range of detection. These data determine the optimal total protein load to
be 4.0 4.0
ng ng
or less for accurate
71737
quantification
86488
of protein expression.

8.0 ng 79795 172976 Protein Load

16.0 ng 75830 345952

Note: The linear range of detection should be determined for both the target protein and loading control protein. This is to
determine the optimal amount of total protein to load to ensure accurate detection of both proteins. This can be performed by
running duplicate samples (e.g. ABCD, ABCD) and cutting the blot vertically to separate the samples. One blot is then used to
determine the linear range of the loading control, while the other is used for the target protein.
Linear
Density

Response
Measured
3 Density Learn more | novusbio.com 2
What to Consider When Using
a Loading Control Antibody

Concentration Dependence and Expression Consistency

Accurate normalization requires that the protein of interest and loading control protein to vary equally with sample
concentration. If unequal variation of one measurement occurs independent of concentration, then normalization fails to bring
target protein expression into accurate proportion with the loading control protein. In this case, expression differences between
samples can be the result of differences in total protein or loading control abundance and not the result of real differences in
target protein expression. Thus, it is crucial to choose a loading control protein with ubiquitous and unchanging expression.

For example, hypoxic conditions have been shown to upregulate the expression of GAPDH, a common whole cell loading control
protein. For this reason, GAPDH is not recommended as a loading control protein in oxygen related studies since its expression
is subject to change as a result of experimental conditions. In addition to normalizing samples via loading controls, minimizing
sources of experimental variability will improve reproducibility, resulting in more accurate quantification of target protein
expression by Western blot.

The Expression Level of the Loading Control Protein Changes in Response to Experimental Treatment. If large
changes in loading control expression are observed despite loading equal amounts of sample on the gel, then a
different loading control protein should be considered. This is exemplified in the image shown above where treatments 1,
2, and 3 result in varying levels of expression of the loading control. Changes in loading control expression due to
experimental conditions will affect normalization and result in inaccurate data analysis. Choose a loading control protein
whose expression remains constant across all experimental conditions.

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 What to Consider When Using
a Loading Control Antibody

Stripping and Reprobing

A common method to determine expression of a loading control protein is stripping and reprobing. Following detection of the
protein of interest, the membrane is stripped of antibodies, then reprobed with a loading control specific antibody. In this
method, it is important that all antibody is stripped from the membrane to ensure generation of accurate loading control
expression data. Residual antibody on the blot may result in artifacts which may impact loading control expression data.
Moreover, blot stripping can result in significant loss of sample protein from the membrane, which could impact expression data
and normalization calculations. After stripping, but before reprobing, the membrane should be tested to verify the first set of
antibodies was completely removed. To confirm complete antibody removal, the membrane should be washed, blocked, and
then stained with secondary antibody. If stripping was complete, then the secondary antibody will remain unbound and produce
no detection signal. If signal is detected, then the stripping conditions must be optimized. Often small changes in incubation
time or temperature are sufficient to remove the remaining antibody. However, a stronger stripping buffer may be necessary in
certain cases for antibodies which are difficult to remove.

UNDER-STRIPPING
UNDER-STRIPPING OVER-STRIPPING
OVER-STRIPPING
BEFORE AFTER BEFORE AFTER
BEFORE AFTER
STRIPPING BEFORE
STRIPPING AFTER
STRIPPING STRIPPING
STRIPPING STRIPPING STRIPPING STRIPPING

RESULT RESULT
Residual antibody on Loss of protein
the membrane results from the membrane
in signal as a result of as a result
incomplete stripping of over-stripping
Blot stripped and Blot stripped and
reprobed with reprobed with
HRP-conjugated primary and
secondary antibody secondary antibody

Insufficient Stripping: A primary antibody was used Excessive Stripping: A primary antibody was used
to probe expression of a target protein. to probe expression of a target protein. Following
Following target protein detection, the blot was target protein detection, the blot was stripped to
stripped to remove antibodies, and then reprobed remove antibodies, and then reprobed with the
with HRP-conjugated secondary antibody to confirm same target protein-specific antibody. The lower
complete stripping. The signal generated post- signal generated post-stripping demonstrates
stripping demonstrates the stripping process was protein sample was stripped from the membrane.
incomplete. Residual antibody left on the Unequal stripping of protein sample across lanes
membrane bound the HRP conjugated secondary can result in inaccurate normalization. Special care
antibody, producing chemiluminescent signal. should be taken when stripping blots consisting of
samples from multiple sample types.

Note: See page 7 for a general stripping and reprobing protocol.

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Summary: Attributes of an
Effective Loading Control Protein

Tips for Choosing the Best Loading Control:

Detection Size Choose a loading control that can be e.g.  If probing for the expression of the

distinguished in MW from your protein autophagosome marker, LC3B (~14-17 kDa),

of interest. avoid using loading controls such as Cofilin or

Cyclophilin B (~20 kDa), since their molecular

weight is similiar to the protein of interest.

Expression Consistency Choose a loading control that is e.g. Cellular stress has been demonstrated to

constitutively and ubiquitously expressed. upregulate HSP60 expression. Thus, HSP60

The expression should not change with should be avoided as a control in cellular stress

experimental treatment, cell type, or studies, as its expression may differ in stressed

tissue type. versus control samples.

Expression Level Choose a loading control that is highly e.g. The nuclear loading control, PCNA, is

expressed in your sample. Common expressed during DNA S phase. Thus, it is not

loading controls are highly expressed genes recommended as a control for

required for basic cellular processes and non-proliferating cells as its expression is

vitality, also known as housekeeping genes. down-regulated in non-proliferating cells.

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 Probing Loading Control Expression
PROTOCOL
Method 1: Stripping and Reprobing

IMAGES FOR PROTOCOL SECTION

Stripping refers to the method of removing primary and secondary antibodies from a western blot membrane. A single blot can
be analyzed sequentially with multiple antibodies by stripping one antibody from the blot and subsequently incubating with an
another antibody. Membrane stripping is often performed as a means to assess the expression of loading control proteins.
Following detection of the protein of interest, a membrane is stripped and reprobed with a loading control antibody to ensure
equal expression of the loading control. This practice is also useful when sample is limited.

Step 1 Step 2 Step 3

Protein of Interest

Loading Control

Probe the membrane with protein Strip first set of antibodies Reprobe the membrane with
of interest specific antibody from the membrane loading control specific antibody

Protocol Protocol
Stripping with heat and detergent protocol Stripping with acidic pH protocol

1. In a fume hood, agitate the blot in  1. Agitate the blot in stripping buffer #2 
stripping buffer #1 for 30 minutes at 50˚C for 30 minutes at 50˚C

2. Agitate the blot in 1X PBS for 10 minutes at room 2. Agitate the blot in 1X PBS for 10 minutes at room
temperature. Repeat with fresh buffer. temperature. Repeat with fresh buffer.

3. Proceed to the blocking step of the immunoblotting 3. Proceed to the blocking step of the immunoblotting
protocol to reprobe the blot with another antibody. protocol to reprobe the blot with another antibody.

Stripping Buffer #1 Stripping Buffer #2

Components: Components:
100 mM 2-mercaptoethanol 25 mM glycine HCl
2% SDS 1% SDS
62.5 mM Tris-HCl Adjust pH to 2
Adjust pH to 6.7

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Probing Loading Control Expression
PROTOCOL
Method 2: Cutting the Membrane

Another common method to probe loading control expression is to cut the membrane horizontally following transfer. Using
this method, protein of interest and loading control proteins can be separated and probed independently with different
antibodies. The method requires a significant difference in size (kDa) between the protein of interest and loading control.
Prestained markers should be used to determine molecular weight and the right location to cut the membrane.

Protocol Step 1 Transfer samples from

gel to membrane
Cutting the membrane to probe the expression
of two proteins at the same time
Cathode (-)

1. After transfer from gel to membrane, carefully

Filter paper
disassemble the transfer stack and rinse the
Gel
membrane briefly in distilled water or 1X TBST. Membrane
Filter paper
2. Gently mark the molecular weight ladder bands Anode (+)
for size detection.

3. Following membrane blocking, use scissors or a

Step 2 Cut the membrane horizontally to separate
sharp scalpel to horizontally cut the membrane the loading control and protein of interest
taking care to avoid cutting too close to the proteins
of interest. Use rounded tweezers when handling the kDa

membrane. Protein
250
of interest 150
(110 kDa) Predicted Size
100
75
CUT HERE
Considerations Loading 50 Predicted Size
Control 37
(50 kDa)
• This method requires a significant difference in 25
20
size (kDa) between the protein of interest and 15
10
loading control protein to ensure clean separation.

• A prestained marker or ladder should be used to Step 3 Probe one membrane for loading
control expression and the other for protein
determine the molecular weight and correct location of interest expression
to cut the membrane.
kDa
Blot 1
250 Probe this blot for the
• Prior to using this method, every antibody 150
protein of interest
100
should be tested on the whole blot to determine 75

antibody specificity. 50

37 Blot 2
25 Probe this blot for
20
15
the loading control
10

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