Western Blot Doctor™ — Protein Transfer Issues

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Overview

The Western Blot Doctor is a self-help guide that enables you to troubleshoot your western blotting problems. In this section, you can find solutions to issues related to protein transfer.

Other sections in the Western Blot Doctor:

Problems and Solutions

Click on the thumbnail that is most representative of your own blot to discover the probable causes and find specific solutions to the problem.

   
 

 

Problem: Air Bubbles in Blot

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A blot with air bubbles – Western Blot Doctor - Protein Transfer Issues

Likely Cause and Recommended Solutions Improper assembly of the transfer sandwich
  • Use more transfer buffer when assembling the transfer sandwich
  • Make sure to roll out any air bubbles trapped between the gel and membrane while assembling the sandwich
  • Stain the blot with Ponceau S to check the transfer quality before proceeding to antibody incubation
Tips If you observe air bubbles on your blots, greater care must be taken to assemble the transfer sandwich correctly. Make sure that the gel and blotting material are well saturated and equilibrated with buffer, and use a glass rod to roll out any air bubbles between the layers at each assembly step. When using Bio-Rad rapid transfer packs with the Trans-Blot® Turbo™ transfer system, it is not necessary to equilibrate the gel with the buffer, and bubbles are conveniently removed using the included blot roller.
 

Problem: Over-transfer

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An over-transferred blot with no signal – Western Blot Doctor - Protein Transfer Issues

Likely Causes and Recommended Solutions Transfer time too long
  • Reduce the transfer time
Transfer electric field too strong
  • Reduce the transfer voltage or current
Transfer buffer not optimized
  • Decrease SDS concentration during transfer by equilibrating the gel in the transfer buffer for about 20 min before sandwich assembly to remove remaining SDS from the gel and avoid over-transfer of small proteins
  • Increase the methanol concentration in transfer buffer up to 40%
Improper gel selection
  • Use higher percentage acrylamide gels
  • Use a Tris/tricine gel when the target protein size is less than 10 kDUse a 0.2 µm membrane to check for over-transfer of small proteins
Large membrane pore size allows passage of small proteins
  • Use a 0.2 µm membrane to check for over-transfer of small proteins
Tips If you observe little or no signal, the most likely cause is sample over-transfer; trials with shorter transfer times and lower transfer voltages and/or currents should be performed. The selection of the proper gel buffer type and acrylamide percentage for your proteins of interest is crucial, and the removal of residual SDS from the gel greatly reduces the over-transfer of small proteins. Increasing the methanol concentration of the buffer increases its polarity, aiding in the removal of residual SDS from proteins. Other problems unrelated to protein transfer may result in weak or no signal detection on a western blot.
 

Problem: Under-transfer

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An under-transferred blot – Western Blot Doctor - Protein Transfer Issues

 Likely Causes and Recommended Solutions Transfer time too short
  • Increase the transfer time if using tank transfer
Transfer electric field too weak
  • Increase the transfer voltage or current
Transfer buffer not optimized
  • Add up to 0.05% SDS to the transfer buffer
  • Decrease the methanol concentration in the transfer buffer to 5% or less• Use lower percentage acrylamide gels
Improper gel selection
  • Use lower percentage acrylamide gels
Tips For large protein transfer, please select a low % gel, for example, 7.5% or 4–15% gradient gels and add .05% SDS to the transfer buffer.
 

Problem: Uneven Transfer

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An unevenly transferred blot – Western Blot Doctor - Protein Transfer Issues

Likely Causes and Recommended Solutions Membrane partially dried or uneven hydrated membrane
  • Make sure the whole membrane is soaked and equilibrated in transfer buffer
  • PVDF membrane needs prewetting in 100% methanol prior to equilibration in transfer buffer
Hardware problems
  • Check the wire connections on the tank transfer unit
  • Make sure the metal plates are clean and have no physical damage; even minor bending of the plate can change the electrical field strength significantly in semi-dry systems
Tips If you observe weak signal over significant areas of the membrane, physical protein transfer problems are the most likely cause. Ensure that the transfer stack and membrane are wetted and equilibrated with buffer (with prewetting in MeOH for PVDF membranes) at all times during transfer, and carefully inspect the transfer apparatus for connection problems.
 
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