If protein has MW <10,000, it may have 揵lown through?the membrane. Reduce transfer time, or place two sheets of transfer membrane soaked in Anode Buffer II under gel in transfer stack, or select membrane with smaller pore size
Isoelectric point of protein is <9
Use alternate buffer system (such as CAPS buffer pH 10.5) with a higher pH
Assemble transfer stack with additional membrane soaked in cathode buffer on the cathode side of gel. This will capture proteins that migrate towards the cathode. Identify membrane location in stack and immunostain all membranes in stack
Methanol concentration
Higher methanol concentration increases binding to membrane but may retard transfer from gel
SDS concentration
Adding 0.005 - 0.01% SDS to cathode buffer can increase transfer efficiency of protein from gel Note: Increased SDS concentration may interfere with binding to membrane
Thick gel
Thicker gels or higher MW proteins may require longer transfer times
Problem: High Background Staining
Possible Source
Test or Action
Membrane does not wet uniformly
Pre-wet membrane in 100% methanol
Inadequate blocking
Optimize the blocking step by trying alternate blocking solutions (e.g., non-fat milk, gelatin, etc.) or increasing time and temperature of blocking step
Cross-reactivity of antibody reagents
Check for cross-reactivity of antibody reagents to the blocking protein
Problem: Low Protein Binding
Possible Source
Test or Action
Overwashing
Keep the length of TTBS washes to a minimum
See 揚oor Transfer?problem for alternatives
Problem: High Protein Binding, Low Signal
Possible Source
Test or Action
Inadequate antibody staining
Check antibody dilutions and expiration dates
Inactive AP conjugate and/or substrate
Add enzyme conjugate to substrate reagents as prepared in step 7 of immunostaining procedure. If color occurs, reagents are performing properly
Poor sample
If positive control worked, sample may not contain protein of interest or it may be present at concentrations too low to detect
Problem: Several Bands are Stained
Possible Source
Test or Action
Non-specific binding of secondary reagents
Check non-specific binding by running extra sample lane and omitting 1?antibody from immunostaining procedure, run with a non-specific antibody control in place of 1?antibody, or perform dot blot and omit the 1?antibody. If staining occurs, choose an alternate 2?antibody
