What happens to the trypsin/proteases used in SDS page in gel digestion? Does it effect on any further experiments, such as rerunning sample on another gel or mass spect?
The added protease will be autodegraded during digestion (as in: cleaved by itself). It is common that you observe trypsin peaks in your sample. These are basically contaminating peptides originating from your added trypsin. If you do a database search and you include the porcine proteome (assuming you use porcine trypsin, which is common), you will see porcine trypsin on your list of identified proteins in all of your samples.
I am not sure what you mean by: "does it have an effect on other experiments such as rerunning the sample on another gel". Do you want to do another SDS-PAGE of your digest? You can (if you use an appropriate gel suitable for peptides), but do know that you will also have trypsin peptides in your sample.
See the added link for a comprehensive list of contaminants in mass spectrometry, among which you will find trypsin peptides.
http://www.pennstatehershey.org/c/document_library/get_file?uuid=67335431-8f0b-4a77-96c0-e5625f40e119&groupId=2384708
Dear Dar
Using of trypsin is a necessary step before performing of MS.
However, for rerunning of digested protein by PAGE, it gives no valuable results because the certain protein is digested partially or completely by proteases and produce several peptides with different Mw.
It can not be conclusded any results from digested peptides on PAGE.
I am agree with Alexander contamination with peptides will be remain and this will affect the data not on the running sample.
sorry i just want that I agree with what postulated by Dr. Alexander. Sorry for the English mistake.
Thank you very much for your replies and answers. I should clarify what I mean by rerunning my sample. I want to look at how many unknown proteins are attaching to a particular sample and to try to figure out how many different proteins. I'm running SDS page before performing Mass Spect. I want to run my samples on SDS page and then cut my band out and do an in gel digestion. Then run my sample again on a different concentrated gel to be positive that I only have one protein present in that band( not several of very similar mw). Since posting this question i have thought about using either 2d gel or 3d gels to separating the samples out further.
Dara,
I am still not sure I understand what you mean. You do in-gel digestion and then mass spec. Do you then want to rerun your digested sample, or do you want to rerun your original sample?
I wouldn't advise rerunning your digested sample as it doesn't contain any intact proteins anymore. If you want to rerun your original sample, there is of course no effect of the trypsin as you haven't added it yet.
Using a 2D gel for what you want to do is best I think. Although, probably your current experiment will already yield you a list of the proteins in your selected band (most likely there will be several proteins unless you performed extensive purification of your sample).
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