Quantitative Western blotting Flashcards
What is a quantitative Western blot?
There are experiments designed to find out if an
antigen is expressed or not in a sample, for example: is
a recombinant protein expressed after induction? If
the answer is yes or no then the Western is qualitative
- Most Western blot experiments however try to detect
and measure relative changes in antigen expression
between samples, for example: “the amount of
protein X is 2.5 times more in treated cells compared
to untreated control cells”. In such case the Western
blot is quantitative.
Why is Performing quantitative western blot is not
trivial?
Quantitative Western blot requires validation of
conditions, reagents (Ab), buffers, careful performance
including normalization, replications to provide
confidence in the results and reproducibility
- The key is to achieve accuracy and precision with as
low as possible variability in order to get statistically
significant results with high reproducibility - These requirements led to more rigorous standards
for publishing Western blot results in peer review
journals
What are Quantitative Western blot general
requirements?
Confirmation that the signal is proportional to the
amount of loaded protein
- Proteins of interest and the loading controls are
quantified within the linear range of detection - Normalization of the signal from protein of interest
against internal loading control, for example:
house keeping proteins or total protein loading - Variability should be kept low to maintain
reproducibility
Determining the linear range of detection?
Dynamic range: the range of intensities that
detection instrument can measure in a single
capture
- Linear range: the range of signal intensities
recorded by the detector that shows a linear
relationship with the amount of protein on the
membrane
What is signal saturation?
- With saturation relative signal intensity does
not increase – reaches plateau - It can be caused by: membrane saturation
(IMPORTANT: do not overload the
gel/membrane); X-Ray film saturation: film has
narrow linear dynamic range 4-10 fold, which
can be found in serial dilutions experiment - Detector saturation – use detection system with
wide linear dynamic range
Define Sensitivity in this case
Sensitivity is the lower limit of detection system. A
detection system with increased sensitivity has extended
lower limit of linear dynamic range
- Blocking buffer reduces background of detection thus
increasing sensitivity (signal/noise ratio) - One might be able to see very weak band (at the limit of
detection, LOD) and not been able to reproducibly
quantify it. Limit of quantitation, (LOQ) is the limit of signal
that can be reproducibly quantified. The rule thumb is
that the value of LOQ is at least 2 standard deviations
above LOD.
Determine the linear range of detection
Linear range of detection of target and the internal loading
control are determined by Western on serial dilutions of
the sample (usually 8-10, 2-fold dilutions)
- The linear ranges of target and internal loading control are
compared to determine the amount of sample to be
loaded to produce linear response from both - If target protein and loading control (i.e. house keeping
genes that are strongly expressed) are expressed at very
different level, it becomes critical to detect both within the
linear range of the signal or normalization will not be
accurate
Explain normalization corrects for variability
Corrects for unavoidable sample-to-sample and laneto-lane variations by comparing signals from target
protein and an internal control in an individual sample
- Without normalization one will not know if changes in
detected band intensities of the target protein reflect
biological change or variability in sample preparation
and transfer - Loading equal amount of protein on electrophoresis is
not sufficient because of loading inconsistencies and
transfer variability
What are the loading control requirements?
Choose and validate appropriate internal
loading control: usually endogenous proteins
used to indicate sample concentration
- The loading control must be unaffected by the
experimental conditions - Must be detected within the same linear
range as the target - Does not interfere with target detection
Total protein as an internal loading control (ILC)
Total protein staining as an ILC provides a robust and
reliable assessment of sample loading. It is becoming the
“gold standard” for Western blot normalization.
- After transfer, but before blocking, the membrane is
stained for protein across the blot. The stained proteins
per sample lane are quantified and the value is used for
normalization. Error and variability are minimized due to
integrated signal from many different proteins in the
sample - Another benefit from total protein staining is that it also
shows inconsistencies of the transferring process
Explain how housekeeping proteins as internal loading
control
HKP are widely used for normalization. It is generally
assumed that they have stable level of expression in
variety of normal and treated samples. However lots
of studies have shown that their expression is not
stable in cells under different treatments, so
normalization using them can change the
interpretation of the results.
* Before using a HKP for normalization it is critical to
verify that its expression is constant across the
samples and the experimental conditions used.
Experimental protocol for normalization by total protein stain
- Loaded sample amount must be in the linear range of
detection, verified in preliminary experiments - Replicated samples must be used to assess variability
- Uniform sample volume across the gel must be used
- After transfer the membrane is stained with one of the total
protein stains. The total signal in each lane is then quantified. - Immunodetection of the target is performed and the signal is
quantified. - Normalization calculations and statistical analysis of replicates
is then performed
How do you interpret data?
Use the normalized target protein values for
relative comparison of the samples. In this
example target protein in UV treated cells is
14% lower.
- Low %CV indicates low signal variability and
high measurement precision. In general the
change in band intensity is meaningful only if
it is at least 2X greater than the %CV
How do you interpret data? Part 2
For a 20% difference between samples (0.8 or 1.2
fold change in band intensity) %CV for replicate
samples should be 10% or less
- %CV of target protein should not increase after
normalization. The purpose of normalization is to
reduce the variability between replicate samples.
A large increase in %CV after normalization is a
sign that the normalization method is not robust
and may be a source of error itself.
Example quantitation with replicate samples
QWB has to answer the question: does the
abundance of a target protein change in a
group of samples? Is there a real difference
between the control and treated samples or
the observed difference is just a noise?
- Such questions cannot be answered without
replicas
