Hello researchgate family,
My name is Trung. At the moment, I am working on a discovery based proteomics project to identify phosphopeptides. And I am using a ziptip protocol adapted from Millipore procedure (in the attachment). It seems to work better than the one I used to work with. So, I am just wondering if anyone know an optimal ziptip protocol for discovery based peptide elution. Thank you so much.
Hi Trung,
i use the following protocol:
materials:
Acetonetril, 0.1% TFA, C18 or C4 Zip Tips
method
equilibration with Acetonitril (reapeat 4 times)
washing with TFA (0.1%) repeat 4 times
loading sample ( repeat 5-10 times)
washing with TFA (0.1%) 4 times
elution with ACN:TFA (50:50)
Desalting by Empore C18 silica packed stage tips
Procedure:
1. Packing two disks of Empore 3M C18 (47mm, 2215 ) material into 250ul pipette tips
2. Wash and equilibrate StageTip by passing through sequentially 20ul of methanol (J. T. Baker, 9093), 20ul of 50% acetonitrile (J. T. Baker, 9017-03), 0.5% acetic acid (HAcO, J. T. Baker, 9508-03) and twice of 20ul of 0.1% TFA (trifluoroacetic acid, Themo scientific, PI28904),
I would advise against eluting with 80% ACN as you end up with a lot of stuff that isn't peptides on your column. The maximum you should go is 50-60% ACN in our experience to have a cleaner peptide solution.
Thanks Tannveer. We also use maximum 60% of ACN for eluting.
It works just fine for our experiments.
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