I have been regularly using SILAC membrane lysis buffer regularly to perform cell lysis in the iTRAQ based quantitative proteomic experiment. I wish to use RIPA buffer and I don't find any potential interfering component against isobaric reagent. I am more concerned about PEG as an impurity in this buffer. If it is there it will give background noise I believe, is that correct?
Yes, PEG is a problem for the MS, it stick to the C18 in the RP separation, but if you are doing some fractionation with a SCX column that should be OK.
I rather not use PEG.
The tris is a problem because it has a amine group, where the iTRAQ reacts.
My suggestion is avoid these, good luck with this
Yu know the problem is, my protein is human forskin sample lysed in buffer with 8molar urea and i am feeling if i use this buffer it will prohibit isobaric tagging. How to change the lysis buffer without changing or compromising with the quality of proteins. I want to put them in a buffer most compatible with itraq reagents.
I think if you use the 8M urea with only a buffer agent such as HEPES at the pH that you required will be fine to digest and later do the labelling with the iTRAQ. I have done this before and is fine as long as you remember to dilute the urea below 2M with the same buffer.
Good luck
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