Troubleshooting Issues in Western Blot Process

Sample, Transfer, and Blocking Problems Identified

In our previous videos, we addressed potential problems in samples, transfer conditions, and blocking methods. These issues might result in a lack of signal or faint bands.

Transfer Conditions Issue

  • Improper Transfer Conditions: This could lead to the target protein not transferring or over-transferring.
    • For high molecular weight proteins, adjust transfer time and include SDS.
    • For small molecular weight proteins, avoid adding SDS but maintain methanol concentration at 20%.
    • Consider overlaying membranes for exceptionally small proteins to prevent over-transfer.

Membrane Treatment Problem

  • Unactivated Membrane or Inappropriate Pore Size Selection: Activate PVDF membranes using methanol and choose appropriate pore sizes based on protein molecular weight.
    • Use a 0.2-micron pore size for proteins less than 20kDa, and 0.45-micron for larger proteins.

Blocking Issues

  • Excessive or Inappropriate Blocking Solution: Address by adjusting protein concentration, shortening blocking time, or substituting the blocking solution.

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