Can anyone suggest to me how to deplete albumin protein from cocoa as this protein is so dominant in my 2D gel that the proteins are getting masked because of it. Please suggest?
You have 2 types of kit for albumin removal : antibody based and Cibacron Blue dye based system.
I do not know how the human directed antibodies crossreact with your modele, even for the dye (often writen as human specific). In collaboration, I have test in horse and canine some antibodies based kit. And they work well.
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Thank you so much for the valuable answers.I am just wondering that whether these kits will be helpful or not for removal of albumin which is plant based as I am dealing with cocoa protein.
While albumin removal by using these kits have you seen any negative impact on other proteins that is there in your sample or not.
Yes and No. You could have some adverse effects on others proteins as albumin is a carrier of various factors. But we increase the detection of the remaining proteins. We could see some proteins with pI and MW near the albumin track.
In fact, I prefer speaking about 2 compartments : the one linked to albumin and the other not linked to albumin.
I think these kits are valuable for 2D gel.
Hi Neha, though I have never tried it with plant proteins, perhaps a technique we regularly use in our 2D gel preparations might help you: it's called ProteoMiner from Bio-Rad and it's used to decrease the concentrations of all high abundant proteins in your sample while retaining all low abundant ones in their exact concentration, even including the ones that are bound to albumin. It's based on affinity chromatography using hexapeptides as ligand-binding sites. Have a look at some of my publications where we used it in. Good luck!
In order to find out that the commercial albumin depletion kits (usually used for human albumin) will work for your cocoa sample look at sequence similarities. see paper below : Primary Structure of the Abundant Seed Albumin of Theobroma cacao by Mass Spectrometry, J. Agric. Food Chem., 2000, 48 (11), pp 5593–5599.
Good luck !
If you have access to larger quantities (1-10mg) of pure antibodies (without BSA as stabilizer etc) against cacao albumin(s), consider immobilizing them on NHS activated Sepaharose to make your own affinity columns at 1..5mg antibody per ml Sepharose. Usually, when using clear, filtered lysates and regenerating the columns by eluting affinity bound material with acidic buffers, these columns may be used "almost forever".
You can use the Affi-gel blue-protein A kit that remove albomin efficiently from your samples. This kit is commercially available , ready to use and its using is very simple.
I used it for some reseraches and found valuable results.
Affi-gel blue-protein- How much it costs and its source of availability
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