Help with iTRAQ-SCX protocol - (Feb/27/2012 )
I am planning to do iTRAQ-SCX followd by LC-MS/MS in one of my experiments. Since I am new to proteomics, I am facing some basic technical difficulties. I have decided to foloow the protocol give in Nature Protocol exchange. I would be extremely happy if you could help me with this.
1. One of my basic question is that whether it is possible to separate SCX part and the LC-MS/MS part. We do not have LC-MS/MS facility and hence I want to out-source the samples. So is it make sense to follow the procedure till the end of SCX-HPLC part and then send the samples out for LC-MS/MS? If yes, in what form will it be - I guess it will be in powder form, especially after the speedVac step.
To this questions one of my friend in another forum replied: "Sure, i think it is fine to separate the SCX from the LC-MSMS part. Yes, just dry the samples in a speedvac or lyophilizer first. You probably will lose IDs by drying the sample completely. But you dont really have any other options. And I think you should still get ok results."
I thank him for his reply. This surely helped.
2. I have decided to do SCX in my lab using PolySULFOETHYL-A from PolyLC company. We have Shimadzu HPLC. Can this specific SCX column be used with the instrument that I have?
3. This question might sound like very kiddish. But I am too new to HPLC. So please bear with me!
How should I collect the fractions after SCX? I know that there are fraction collectors available like Probot fraction collector. But is this absolutely necessary for SCX? Or can we manually collect the fractions and if yes, into what will we collect? Probably into an microcentrifuge tube?
I request everyone to participate in the discussion and help me solve these problems with your insights and suggestions.
Thank you very much in advance!
Thank you so much and sorry for a very late reply!
I was wondering if someone could provide any iTRAQ-tested cell lysis buffers? From an online research I found that 1 M triethylammonium bicarbonate (TEAB) with up to 0.1% (wt/vol) SDS is a good choice. How about RIPA buffer?
What I am using right now (for experiments other than iTRAQ - such as western blotting etc.) is a buffer with:
Is this going to interfere with iTRAQ steps??
Thanks a lot!