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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.
|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, PositionClick for more troubleshooting tips