question about the SAXtip protocol

[cavaliersun2003]

Hello!
I'm wondering if anyone have the experience of using the SAX StageTip sample preparation protocol described in http://www.ncbi.nlm.nih.gov/pubmed/19848406 (Wisniewski et al, J Proteome Research, 2009)? and http://www.ncbi.nlm.nih.gov/pubmed/19377485

I have tried later one several times, but didn't get nice fractionation at all, briefly, my fraction at pH 12 (flow-through) and pH 11 contains lots of peptides, while the sequential step elution at pH8, 6, 5, 4 only hit very few peptides, and pH2 (also contains 200mM NaCL) contains lots of peptides too.

Does anyone have suggestions?

There were actually lots of protocols with different modifications, some use different Britton–Robinson buffer (0.02M vs 0.04M, different pH steps), and making the tips in different ways (3 layer vs 6 layers). I'm wondering which one is better. Also, how mucn peptide you can load onto a SAXtip?

thank you!

[zougman]

Hi,
I have just done a simple experiment for the peptide-load-vs-SAX-plugs-number question - you can see the attached image.
Loading 70 μg of a cellular digest results in the no visible peptide flow through the 4-plug SAX tip (17-gauge plugs), whereas the flow-through is visible in the 1-, 2- and 3-plugs tips.
I think preparing 3 or 4 SAX fractions should be well enough to cover the most of a proteome providing you have a satisfactory digest methodology and access to the QE-type of instruments.

AZ