We want to remove the high abundance proteins in plasma by using 'Pierce Top 12 Abundant Protein Depletion Spin Columns' so that we can use this plasma for 2D-DIGE. Thermo Fisher advises max 10 µl plasma / column, has anyone tried to add more than 10 µl (for example 20 µl)? Thank you in advance!
I have used an albumin and IgG depletion column from BioRad. But what I have to say may help you. In 10µl of plasma you dont know the protein amount. If the protein concentration is high you may be overloading the depletion column.
I recommend you to quantify the protein sample first and then standarize the amount to load in the column. With my columns I used 4mg of protein sample and it worked very well
Hi Maaike,
We have used this type of columns recently, both with serum and plasma. In both cases we followed the vendor´s instructions, so we used 10 uL of sample volume.
With serum we have observed that six out of the twelve depleted proteins were identified among the 30 most abundant proteins in our study (A2MG, ALBU, TRFE, HPT, A1AT, APOA1). APOB was the most abundant protein, and several complement not depleted by the kit were also among the most abundant proteins identified in our samples (CO3, CO4A, CO4B, CFAH, CFAB). All this data is coming from a small study with n=15, and based in spectral matches. Overall, with plasma we observed quite similar results (n=35).
Therefore, I would not load the column more than what is recommended, since you would have undesired effects. As Nancy mentions above, loading less one could expect more efficient depletion rates, which could be enough for further digestion and shotgun analyses.
Best,
Felix
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