I conducted a SDS PAGE of my protein sample and I'm expecting three different subunits at around 20 kDa. However, I only observed a thick band around the said MW while i still observed all the corresponding protein bands for the MW markers.I prepare fresh reagents except that I still used an old sample buffer. It is possible that the sample buffer is the culprit for the said observation?
Hi,
Old sample buffer could be one of the suspects. Usually the mercaptoethanol, which is necessary for reducing the sample disintegrates over time. This you can make out by the lack of odour of the sample buffer. You may have to spike it again and try, or better prepare fresh sample buffer.
Also pH of the sample buffer is becomes acidic over time. This can be observed by the change in colour of the sample buffer (bromophenol blue usually turns green in acidic condition, but blue when basic). Try spiking a small amount of say tris pH 10.0 to make it basic again.
However, it is unclear from what you have written if the other factors are in order, such as gel percent, since you are expecting 3 bands around 20 kDa. There may have not been adequate separation to distinguish all the 3 bands. What % of gel did you use?
Santosh
Dear Bong
- I doubt it is the sample buffer: That is generally stable
- Are you using the same protein lysate and if kept @ -20C this can start to degrade if more than 6 months old and or with freeze thawing, even on ice
- Accordingly, if from this lysate you have observed the three bands up similar SDS PAGE conditions when the lysate was fresh, then throw the old lysate away and make a fresh lysate to WB
- If however, the lysate is fresh and/or you have never been able to resolve your 3 bands approximating to 20kD then the problem could be your electrophoresis conditions and in particular the % acrylamide in your gel: 20kD is on the small side for a protein so you would require at least a 10% acrylamide gel and if your 3 bands only differ by say 3-5KD then resolving such bands could be tricky without optimising your SDS PAGE: With a gel < 10% it could be your 1 thick band is actually your 3 isoforms but not not resolved or separated
- See for example
- https://www.researchgate.net/post/How_do_I_run_very_low_molecular_weight_proteins_on_an_SDS_PAGE_gel
- https://www.thermofisher.com/uk/en/home/references/protocols/proteins-expression-isolation-and-analysis/sds-page-protocol/one-dimensional-sds-gel-electrophoresis-of-peptides-and-small-proteins-with-pre-cast-gels.html
Thank you santosh and laurence for the suggestions, I will try it later.
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