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Western Blot Troubleshooting Guide

As your Western blotting partner, our technical support team is ready to help you analyze protein expression and protein-protein interactions with sensitivity and reproducibility. Troubleshoot your Westerns using the reference guide below, download the Protein Blotting Handbook (PDF) for principles and protocols, or for customized assistance, visit: www.sigmaaldrich.com/techservice.

Western blotting is a protein detection technique in which mixtures of proteins are first resolved in a polyacrylamide gel in the presence of sodium dodecyl sulfate, so that the protein are separated according to molecular weight. Following electrophoresis, the proteins are transferred and bound to a microporous membrane, and identified using two parameters:

  1. Molecular weight, determined by comparing migration to a ladder of proteins of known molecular weight
  2. Presence of a signal caused by binding to a specific detection antibody

Because this technique involves multiple steps, and because there is no one set of optimal conditions applicable to all samples, all target proteins, and all antibodies, Western blotting researchers often spend countless hours systematically varying conditions and troubleshooting blots to get the best signal-to-noise ratios. Nevertheless, Western blots are still very frequently used. In a recent survey, we found that over 50% of respondents performed Western blots every week, primarily for protein identification. Despite numerous improvements in the technique, Western blot users cited that obtaining good signal-to-noise and reproducibility were their top challenges.

Browse the guide below to determine which symptom may be affecting your Western blot. What you think is a problem with membrane transfer may actually be a cell lysis problem, and vice versa.

Troubleshooting Topics

  1. Preparing Cell Lysates
  2. Western Blot Analysis of Immunoprecipitation (IP-Western)
  3. Sample Preparation & Gel Electrophoresis
  4. Dot/Slot (Filtration) Blotting
  5. Semi-Dry or Tank Electrotransfer
  6. Protein Visualization
  7. Immunodetection