Water for Western Blotting

Impact of Water

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In Western blotting, water is used to prepare the transfer buffer, buffers used to prepare the blocking solution, primary and secondary antibodies, and the wash solutions. To enhance protein detection, it is important to use water that is free of contaminants.

• Bacteria and bacterial by-products
  Biologically active molecules resulting from bacterial contamination will compromise the quality of the results from a Western blotting experiment. For instance, if the secondary antibody is labeled with alkaline phosphatase, the water used in Western blotting needs to be alkaline phosphatase-free to make sure that background chemiluminescence is kept at a minimum. In addition, bacteria can release proteins that could interfere with the blotting process. 
   
• Ions
  To have reproducible results, the ionic strength of the buffers used in Western blotting needs to stay the same from one run to another. The water used to prepare the buffers needs to have consistently low ionic contamination. 

• Organics
  Water that is grossly contaminated with organics could affect fluorescence signals. It is advisable to keep organic contamination to a minimum. 

Experimental Results

Comparison of background chemiluminescent signals from different types of water shows that the lowest background signal comes form ultrapure water (Figure 3).



In an experiment where Western blotting was used to detect heat shock protein 70 (HSP70) in a rat liver lysate sample, blots that were processed using ultrapure water from a Milli-Q® system equipped with ultrafilter gave better signals than the blots processed with bottled water (Figures 4A and 4B).

More Information

• Application Note: Bacterial alkaline phosphatase 

Posters

• Impact of Water Quality on Western Blotting
• Variations in chemiluminescence and fluorescence signals due to water quality

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