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