I am a beginner of SILAC based mass spectrometry and I want to know the differential protein expression both my control and experimental cell lines. From various literature studies I think that Spike-in SILAC is best for me. I have designed an overview of the steps -
1. Lysis of cells and protein extraction
2. Protein quantification
3. Mix 1:1 (amount) ratio of proteins both from control and experimental cell lines.
4. Denaturation, alkylation and in-solution digestion of proteins.
5. Clean up.
6. Mass-spec analysis
Is there any other steps required ? I am using Gibco cell culture media and Gibco-FBS. Which kit is best for SILAC? Should I buy reagents separately or a kit?Should I mix only two samples or more than two samples for mass analysis? Please help me.
I found this paper helpful for my SILAC project:
http://stke.sciencemag.org/content/2/72/ra24
and this is my manuscript describing how we did it.:
https://www.ncbi.nlm.nih.gov/pubmed/26200910
Thank you Elizabeth for your reply.Actually, I have planned in solution digestion instead of in-gel digestion. Is it ok ?
Ronald Hay's group in Dundee, Scotland have a number of methods papers and are pioneers when it comes to mass spec. Take a look at their publications as im sure this will answer most your questions
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