what should be the concentration of proteins to start with if one has to label 100ug of proteins with iTRAQ labels since some amount of proteins would be lost after acetone precipitation(during decanting) and during sample processing before labeling.Can anyone suggest any alternative way to acetone precipitation to remove interfering substances before labeling peptides .
Protein loss depends on the complexity of the sample, the volume of acetone and the time of precipitation. Complex samples precipitate better, longer time intervals in acetone improve the yield. I usually precipitate with at least 3 volumes of chilled acetone. Alternatively, you can desalt using PD-10 columns. Just for you to know that there is no magic approach, each of them will result in protein loss. Personally I would recommend acetone precipitation.
Hi Abhinav,
Can you brief us about how you manage to dissolve your protein pellet (obtained after acetone precipitation) ? This would give a better idea to how best can your query be addressed.
In our lab, we use Trizol for protein separation followed by Guanidine Hydrochloride and acetone washes to the pellet. The obtained pellet is dissolved in buffer containing 4M urea, DTT and CHAPS. We then proceed with exchanging the buffer to TEAB (LC-MS compatible) in 3kD cut-off filters. We start with around 100ug of the dissolved protein to get around 70-80ug after buffer exchange and then proceed for iTRAQ labelling.
The above method is circuitous but is of use if the protein is to be used for conventional 2D as well as LC-MS at a later stage.
However, if you were to start with protein extraction from a tissue, it would be worthwhile lysing the same in TEAB buffer with 0.1%SDS and proceeding for iTRAQ labelling to avoid a greater protein loss due to acetone precipitation.
Hope you find the reply helpful.
Regards,
G.Saicharan
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