Dear researchers,
I would like to ask
is it necessary to do Elution of proteins from Polyacrylamide gel before sending the protein sample to Nano-LC-TOF-MS Analysis?
This is what I plan,
I would like to send my samples to LC-MS for analysis after running SDS-PAGE.
But I have no idea whether I want to carry out the elution of protein or not.....
Look forward to your reply.
Thank you.
Hi Jennifer Leong , yes and no.
for MS analysis, if the samples have to be run on SDS-PAGE first, then samples will be digested with trypsin directly in the gel with a method called In-gel digestion, then the tryptic peptides will be cleaned and analyzed by MS.
So, yes the proteins/Peptides has to be eluted from the gel, but no usually its not you who is going to do this step, as in-gel digestion should be in the MS lab by a specialist.
Here is a good article for reference
Hi Jennifer,
I do lots of gel-LC/MS/MS, so I think I can answer your question.
If you want to do MS after running a SDS-PAGE, then you have to first make sure you stain your gel with the appropriate dye to visualise your bands (I use colloidal Coomssie). Then you have to cut your bands out, destain the bands, reduce and alkylate, then tryptic digest your protein.
After tryptic digestion, you'll have to speedvac to concentrate your sample, then acidify your sample for MS.
The paper Rabab G. El Mergawy shared is the paper that I follow and then modify to your own application.
Cheers,
Jennifer
Hi Jennifer,
peptides will be diluted from your gel piece after digestion. As a leader of service group I always prefer to get samples in gel form and apply in-gel digestion protocol by myself. You have to ask people who are going to run MS analysis for you what kind of samples they would like to receive.
In this case I can refer you to my paper where we apply this in-gel method in "high-throughput" way:
Article Hit-Gel: Streamlining in-gel protein digestion for high-thro...
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