Western blot | troubleshooting | samples turn yellow and precipitate

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Why western Blot samples turn yellow and precipitate?

ph drop change in color of Bromophenol blue


  • Sample appears to be acidic. Bromophenol blue in the loading buffer turns to yellow at acidic pH (below 3). Try to adjust the pH by adding NaOH [1].
  •  Reason for that is proteins may be getting degraded due to heat exposure. One researcher had the same problem and so instead of keeping samples at 95°c he started warming at 60-65° C. So anyone can try it out and as [1] said check the pH as well of the loading dye [2]. 
  • Heating a sample also changes pH. An altered pH is not only responsible to turn the sample color yellow, but may also precipitate the proteins. Keeping SDS containing samples in ice is another common mistake to see this precipitation [3]. 
  • Another researcher said that he has got to do more with the total protein concentration in the lysate. He faced the same problem of precipitation upon denaturation at 100°c. He added 1/10th volume of 10% SDS and then boiled. Protein did not precipitate. The high concentration of SDS will solubilize the proteins and prevent their precipitation [4].

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