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tryptic digest in solution

tryptic digest in solution - Mass Spectrometry Forum

tryptic digest in solution - Discuss and post questions about LC-MS, Q-STAR, MALDI-TOF, and other Mass Spectrometry Questions in the Mass Spec Forum.


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Old 06-17-2008, 03:51 PM
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Lightbulb tryptic digest in solution



Hi!

Anyone have a good protocol for tryptic digestion in solution for a pure protein? Are there many differences compared to in-gel digestion?

Do you still use zip-tip clean up afterwards?

Thanks!!

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Old 06-18-2008, 03:26 PM
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Default Re: tryptic digest in solution

Hello Lumena,
as you have came quite far in purifying protein and everything, I would check first with your proteomics sequencing facility. Depending on what machine they use they may ask you to use certain buffers, a limit of SDS concentration and other tips.

Please check with them first (also it is a good idea to ask around if the site is a good one) as I had the same problems when I tried to sequencing some proteins.
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Old 03-02-2009, 09:52 PM
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Default Re: tryptic digest in solution

Hello Lumena:
Mainly it depends on the method of analysis following digestion. Are you doing MALDI then you will need to do sample clean-up using zip-tips. If your are doing HPLC-ESI-ms then no sample clean-up is necessary. One of the main things I have found is using a good tryptic enzyme such as the new recombinent trypsin. Keeping the pH as low as possible (7.5-7.8) and use a time as short as possible. With this enzyme you can increase the enzyme ratio and decrease the digestion time to as little as 30 minutes. Tris buffer I find is still the best.
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