Troubleshooting Guide: Western Blot

Problem: Poor Transfer

Possible Source	Test or Action
Membrane does not wet uniformly	Pre-wet membrane in 100% methanol
MW of protein is less than 10,000	If protein has MW <10,000, it may have �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 �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
Primary antibody is not monospecific	Use a monoclonal or affinity purified antibody