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Western Blot Troubleshooting: Wrong Band Sizes

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

Keywords: Western Blot, Troubleshooting, Unexpected, Band Size, MW, Position

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