Western Blot Doctor™ — Loading Control Issues

print

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 loading controls.

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.

Western blot with varying levels of control proteins – Western Blotting Doctor - Loading Control Issues

Protein level varies among
samples

Western blot with saturated levels of control proteins – Western Blotting Doctor - Loading Control Issues

Protein detection saturated

 

Problem: Protein Level Varies Among Samples

Back to Top

Western blot with varying levels of control proteins – Western Blotting Doctor - Loading Control Issues

Likely Cause and Recommended Solutions Loading control protein expression level changes in response to experimental treatment
  • Identify and validate the consistency of the loading control protein under the experimental conditions
  • Stain the blot with total protein stains and use total protein measurement on the blot as a loading control
Tips If you observe wide variation in the level of signal from the loading controls and are confident that the gel was loaded with the same amounts of protein in each lane, additional measures should be taken to validate signal normalization for each sample.

The use of housekeeping proteins for normalization in western blotting is prone to a number of pitfalls, and the use of total protein normalization as an alternative to
single-protein loading controls has a number of advantages.

Please see the references below for more information.
 

Problem: Protein Detection Saturated

Back to Top

Western blot with saturated levels of control proteins – Western Blotting Doctor - Loading Control Issues

Likely Cause and Recommended Solutions Loading control protein level changes in response to experimental treatment
  • Titrate control loading downward to eliminate saturation
  • Dilute the antibodies or reduce incubation time to avoid saturated detection
  • Select another consistently expressed, low-abundance protein as the loading control
  • Stain the blot with total protein stain and use total protein measurement on the blot as a loading control
Tips If you observe oversaturation in the level of signal from the loading control, the most likely cause is overloading of the control; trials with smaller amounts of controls should be performed. Again, staining and imaging the blot with a total protein stain can allow normalization based on total protein transferred to the blot. Oversaturation problems in general can be addressed by diluting the detection antibodies and/or reducing exposure time; several trials may be necessary to determine the optimum conditions for your samples.

Stain-free technology can also be used a tool to replace traditional loading controls in western blotting. Please see the references for more information.
 
 

References

Publications on total protein normalization as a loading control in western blotting — Bio-Rad bulletin 6349

Publications on the pitfalls of using housekeeping proteins for normalization in western blotting — Bio-Rad bulletin 6350

Publications on using stain-free technology for loading controls in western blotting — Bio-Rad bulletin 6351

Heidebrecht F et al. Improved semiquantitative western blot technique with increased quantification range. J Immunol Methods 345(1–2), 40–8 (2009).

Ask an Expert

Call us at 1-800-4-BIORAD (1-800-424-6723)

Sign Up for Bio-Rad Updates!
Enter your email address below to receive your choice of the latest news, promotions, and more.