Documentation

The Western blot analysis is a powerful tool for protein detection. In standard, qualitative Western blots, the presence or absence of a protein is verified, the molecular weight of the protein is estimated by comparison with known molecular weight markers, and the amount of protein contained in the sample can be estimated by visual analysis. Quantitative Western blots to define the absolute or relative amount of protein in a sample can also be performed using precise methods, sensitive detection reagents and advanced imaging techniques.

Quantitative Westerns are performed in the same manner as qualitative Westerns, however the resulting signal must be captured and quantified. Digital capture and image analysis using the appropriate software allow for the sensitive quantitation of protein amounts.

When not performed accurately, quantitative Western blots can give erroneous results; care must be taken to understand the limits of the assay and the parameters that affect the ability to accurately measure protein quantity.

Read below for factors that can affect quantitative Western blots.

1.Precision

The process of Western blotting can be imprecise. Loading of samples, differences in transfer of protein to the membrane, antibody lots, etc,. all add variability to the procedure. It is important to limit variability as much as possible.

a.Pipetting errors

Be careful when loading samples onto the gel. Pipetting errors can affect the amount of protein in each lane.

b.Standardize incubations

Accurately time all incubations including equilibration of gel in transfer solution, blocking, antibody and substrate incubations.Keep transfer time standard to prevent transfer of proteins through the membrane or incomplete transfer from the gel.

c.Make up all buffers precisely

Buffer quality can affect transfer of proteins.

d.Use wet transfer

Wet transfer provides a higher transfer quality than dry blot systems.

e.Compare within the same blot

Due to transfer and handling differences, only compare proteins on the same blot and not between blots.

f.In-lane normalization

Normalization factors out inconsistencies in protein concentration resulting from loading errors or sample-to-sample variation.

i.Normalize the protein of interest to an internal reference, such as a “housekeeping protein”

ii.Make sure the internal reference does not vary with treatment conditions used in the experiment

iii.In the absence of appropriate endogenous references, lysates can be spiked with a known quantity of protein prior to loading

2.Sensitivity and linear dynamic range

Sensitivity refers to the minimum amount of protein that can be detected and is dependent upon antibody quality, antibody concentration and exposure time.

The linear dynamic range is the range in which signal intensity is proportional to the protein quantity on the blot. Good quality quantitation of a Western blot occurs when the signal is within the linear range.

a.Use good quality antibodies

i.Use a highly sensitive antibody that allows precise quantitation of both strong and weak signals

ii.Use an antibody with a low limit of detection (LOD - amount of protein that can be distinguished from background)

b.Titrate all antibodies

If doing multiplexed Western, titrate each antibody individually prior to combining antibodies.

c.Don’t use excessive amount of protein leading to saturation

d.Precisely time all incubations

e.Decrease exposure times

f.Use a charge-coupled device (CCD) camera-based imager; Never use film

i.CCD has a higher sensitivity and broader linear dynamic

ii.X-ray film can be saturated by signal and give false values

3.Signal stability and decay

Quantitation of the signal can only occur if the signal is stable enough to allow for appropriate imaging times. Results can also be skewed if the signal does not decay in a linear fashion.

a.Use a sensitive substrate with high signal stability such as WesternBright Quantum HRP susbstrate

WesternBright Quantum was developed to provide high sensitivity for low abundance proteins, does not deplete in the presence of abundant proteins, and is stable for hours.

b.Incubate blot with enough substrate to cover the entire blot

c.Time substrate incubation precisely

4.Signal to noise ratio

The signal to noise ratio is a comparison of the level of the specific signal to the level of background noise. High ratios are desired to allow detection of low abundant proteins and increase the dynamic range. Several factors affect the signal to noise ratio.

a.Titrate antibody

Titrating the antibody will ensure that the highest signal is achieved with the lowest background.

b.Optimize blocking

Blocking will decrease the overall background, however care must be taken to not mask the protein being analyzed.

i.Do not “overblock” and mask protein

ii.Use appropriate blocking buffer

1.Try 0.5-1% non-fat dry milk (or even less) instead of commonly used 5%

iii.“Pre-block” less sensitive antibodies

l1.Incubate primary antibody with blocking solution in a small volume prior to incubation with membrane

c.Use CCD device for image capture

d.Read [[high_background_in_western_blots_-_causes_and_solutions|High Background in Western Blots]] for tips on reducing the background

e.If using fluorescent antibodies, use a low fluorescent membrane