Thanks very much for you answer, Christina. To be honest the different informations in the forums were a bit confuse.

May I ask you a couple of think about your advice?

1. If I dissolve the protein pellet in 20ul of 8M Urea and then I diluted until 2M, the final volume will be 80ul, is the increase of the volume going to reduce iTRAQ efficiency? or just adjusting some of the volumes on the protocol (adding 4ul of Cysteine Blocking Reagent instead of 1, for example) will be enough?

2. Can I use TEAB ph 8.5 instead of Hepes? It is just because is the same substance used in the Dissolution Buffer from the kit (by the way, the TEAB in the Dissolution buffer is 0.5M but I've seen many people using 50mM for resuspension, I wonder if there is something wrong in the handbook).

Thanks again for your help!

Hi Daniel

A procedure that is often used is to dissolve in 8M urea, reduce alkylate and digest with LysC- resulting in more soluble peptides. you can then reduce the urea concentration to 2M and perform trypsinisation

regards

Howard

Thanks all for your help. I will do the 8M Urea and then reduce it to 2M. I will try different approaches to achieve this (unfortunately we don't have things like a vacuum concentrator, what is a pity). I may try to dissolve a pellet with the 8M Urea buffer, diluted to 2M and then evaluate protein concentration and use 20ul of this final solution for the iTRAQ protocol. Hopefully doing that I will have at least 50ug of protein per sample (75ug will be optimal).

Anyway,

Thanks to all of you for your advice (I hope will be not necessary to bother you anymore)

You can also try Rapigest, an acid cleavable detergent that does not interfere with trypsin digestion. 0.2% rapigest in 0.05M TEAB (iTRAQ dissolution buffer), boil for 5 mins. After reduction, alkylation and digestion add TFA to pH2, incubate at 37oC for 30 mins then spin at 15,000 x g, and discard the pellet.

" I precipitate between 75-100ug of protein using acetone'' in my opinion, if can you use some other precipitatione solutions

Thanks Kimberley. That seems a good idea too. I assume that the Rapigest detergent in TEAB is added to the pellet after acetone precipitation. Can you explain a bit more why after trypsin digestion I should add the TFA to pH2? if I do that the remain supernatant (that I assume will be used for the iTRAQ labeling) has to be readjusted again to be over 7.5 (that is what the handbook recommend).

Thanks

Yes, you can add this to an acetone or other precipitant. Make sure you don't over dry the precipitated protein as this could contribute to the resolubilization problem. The TFA cleaves the Rapigest, allowing its effective removal by centrifugation. Rapigest is a Waters product, please refer to the product page for more info. At this point I desalt the peptides (HLB column), quant the final product (Qubit, invitrogen), and dry desired aliquots in a centrifuge concentrator (TEAB and TFA are both volatile). Re-suspend in 0.5 M TEAB and your pH should be fine for iTRAQ labelling.