FAQ

The background of a western blot does not always appear clean and flawless. Blotches, streaks, and spots are all common artifacts that can make it hard to interpret and publish your results. These artifacts are most commonly the result of uneven coating of buffer or antibody, the membrane drying out, or aggregates forming in the antibody or blocking buffer. Follow these tips to identify and solve the cause of your imperfect western blot background:

Problem Cause Solution
Blotched background Uneven antibody distribution Agitate during incubation to coat the membrane evenly in incubation buffer
Membrane dried out unevenly Make sure membrane is thoroughly wetted before starting the protocol
Ensure the membrane does not dry out during any step
Uneven wash/incubation buffer coverage Increase volume of wash and incubation buffers
Do not stack membranes during incubation
Flecked background Secondary antibody aggregation Increase secondary antibody dilution to prevent aggregation
Spin down or filter out antibody aggregates
Clumps of blocking buffer binding secondary antibody Use fresh blocking buffer
Increase Tween 20 concentration of blocking buffer
Filter blocking buffer before use
Use a different blocking reagent, such as albumin, BSA, or casein
Wash membrane with wash buffer before antibody incubation
Buffer contamination Mix new buffers
Filter buffers before use
White spots with no protein transfer Air bubbles trapped between gel and membrane during transfer Carefully squeeze out bubbles from between membrane and gel using a sterile glass rod
Use enough buffer to saturate the membrane
Category: faqs

Typically, polyacrylamide gel electrophoresis separates proteins by molecular weight, with large proteins migrating slower than small proteins. This process is can be affected by several factors: protein degradation can cause proteins to appear shorter than the expected length, glycosylation can cause proteins to appear larger than predicted, and nonspecific antibody binding can cause multiple bands to appear on a blot where only one is expected. Use these tips to identify and resolve the source of your unexpected band sizes.

Problem Cause Solution
Bands have higher MW than expected Proteins are glycosylated or bear other post-translational modifications Review literature and identify modified forms of your target protein
Strip post-translational modifications with enzymatic treatment
Bands have much higher MW than expected Protein aggregation Decrease protein concentration
Prepare new sample with fresh loading buffer
Incomplete denaturation or residual disulfide bonding Denature the protein with urea
Use stronger reducing agents
Use fresh 2-mercaptoethanol or DDT to strip disulfide bonds
Bands have lower MW than expected Protein sample has been digested or degraded Use fresh sample from frozen stock
Use a lysis buffer with proteinase inhibitors
Primary antibody is detecting splice variants Review literature to identify splice variants of your protein
Try a different primary antibody
Primary antibody binding a similar epitope on a different protein Run a negative control to detect other proteins that react with your antibody
Multiple bands Primary or secondary antibody contaminated with nonspecific IgG Use Boster primary antibodies guaranteed free of nonspecific IgG
Nonspecific binding of primary antibody Increase antibody dilution
Affinity purify primary antibody to select for only desired binding activity
Use Boster primary antibodies guaranteed to only bind their indicated targets
Nonspecific binding of secondary antibody Reduce antibody concentration
Run a negative control with just the secondary antibody to detect nonspecific binding
Insufficient blocking Use higher concentration blocking buffer
Block for longer
Add tween 20 to blocking buffer
Ionic interactions Increase stringency of washing step
Increase salt concentration of incubation buffers
Include stronger detergents in the washes
Category: faqs

A weak western blot signal is characterized by faint or indistinct bands. While the bands may be barely visible at their predicted sizes, weak signal can often require repeating the experiment. Common sources of this error occur during protein transfer and detection. Use these tips to identify the source of the error and get better results:

 

Transfer issues Insufficient sample concentration Increase the amount of starting material
Concentrate your sample using immunoprecipitation or similar procedure
Transfer too vigorous Reduce transfer time or voltage to prevent small proteins transferring completely through membrane
Use a secondary membrane to capture proteins transferred through the primary membrane
Use a membrane with smaller pore size
Inadequate transfer Increase transfer time or voltage
Sandwich assembly oriented incorrectly Make sure the sandwich assembly is oriented correctly relative to the electric field
Check the polarity of the electric field
Incorrect transfer buffer PH Adjust transfer buffer PH to be 2 points lower than the pI of protein sample to optimize charge:mass ratio
Detection issues Insufficient antibody concentration Use a dot blot assay to optimize protein concentration
Insufficient antibody binding affinity Reduce washing stringency
Increase antibody concentration
Use Boster high affinity primary antibodies
Insufficient sample loading Use more starting material
Concentrate sample prior to loading
Antigens masked by dry milk blocking solution Nonfat dry milk can sometimes mask antigens. Try using a different blocking reagent
Category: faqs