The sample I am using is human plasma. This is my workflow for my pilot study:
1) depleted (albumin and igG) and non depleted sample were used to compare which one give better protein coverage.albumin and igG were depleted using spintrap column from ge healthcare and for non depleted sample,crude plasma was used.
2) 10 ug of protein from each samples then applied for 1d electrophoresis. 1cm of each gel lane was excised (a total of 6 fraction were collected).fractions were then digested using trypsin.
3) each fraction analysed using lcms(nanolc,qexactive orbitrap, proteome discoverer). 6ul of samples volume were injected into lcms machine.
my great pleasure if fellow researchers and experts did not mind to give me advice on my current situation so that i can improve my result...tq...
Dear Zulzikry: Several factors need to be considered: 1. Many proteins loosely associate with albumin, and the removal of the latter can cause a huge loss of countless "small proteins"; 2. One other large amount of protein that can over-shadow your protein profiling is the fibrinogen. So, serum is cleaner than plasma. One has to choose between prior protein removal or over-shadowing, both of which results in your "protein loss". My limited experience told me that use of less sample (say 0.5 to 1 ug rather than 10 ug) may give you a better resolution for direct analysis of SDS-PAGE separated serum (be sure to bMSH-reduce and boil before loading). Good luck.
tq Dr for the advice...i will take your point into consideration upon furthering my optimization of this study...tq very much Dr...
There are several suggestions proposed as the following, you may take them into consideration
1) increase the injection volume; 2) concentrate your sample if the maximum sample volume has been achieved; 3) make sure to clean up the salt in the sample or the salt may inhibite the ionization
thanks zhang for the suggestion...hope it works fine... :)
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