Sigma’s ProteoPrep® Blue Albumin and IgG Depletion Medium contains a combination of 2 media: Blue dye conjugated to an agarose-based matrix and protein G agarose. This combination helps to specifically remove two major abundant proteins of serum: Albumin and IgG, without much non-specific binding to other proteins present in serum. Removal of albumin and IgG allows detection of co-migrating proteins and also allows higher sample load, which improves detection of low abundant proteins. It is better than traditional Cibacron blue resin-based depletion of albumin.
The ProteoPrep® Blue Albumin and IgG Depletion Kit comes with 10 ml of ProteoPrep® Blue Albumin and IgG Depletion Medium, ProteoPrep® Blue Equilibration Buffer, Protein Extraction Reagent Type 4, spin columns and collection tubes which are sufficient to process 25 serum samples. The kit is to be stored at 2-8ºC. The ProteoPrep® medium is supplied as a 60% slurry; it should be kept at room temperature for 20 min before use so that it can be thoroughly resuspended. Following resuspension, the slurry should be aliquoted and kept at –20ºC. ProteoPrep® Blue Equilibration Buffer should be reconstituted in 40 ml of distilled water to make final volume of 50 ml. Protein Extraction Reagent Type 4 should be reconstituted in 15 ml of distilled water to make a final volume of 23 ml. This kit does not require the serum sample to be diluted.
Albumin depletion by this kit is very quick and does not require other reagents. First, the ProteoPrep® Blue Albumin and IgG Depletion Medium is equilibrated. To do this, place 400 ul of the resuspended medium in a spin column and is centrifuge in a collection tube at 10000 rpm for 10 sec to removed the storage buffer. 400 ul of equilibration buffer is then added to the column and it is centrifuged at 10000 rpm for 10 sec; the buffer is removed and the spin column is placed in a new collection tube. 50-100 ul of serum is applied on top of the medium bed in the column and incubated at room temperature for 10 min, then it is centrifuged at 10000 rpm for 1 min. The flow through is added to the column again and incubated for 5 more min and centrifuged at 10000 rpm for 1 min. The flow through is again collected (first eluate). To collect the remaining unbound proteins, 100 ul of equilibration buffer is added to the column and centrifuged at 10000 rpm for 1 min; this flow through is collected and pooled with the first eluate. This combined flow through contains your depleted serum extract. Optionally, bound proteins can be eluted by adding 150 ul of Protein Extraction Reagent Type 4 to the column and centrifuging at 10000 rpm for 1 min. The eluate is collected and another 150 ul of extraction buffer is added and centrifuged at 10000 rpm for 1 min; this eluate is collected and pooled with the previous eluate. This eluate can be analyzed on SDS-PAGE. The albumin and IgG depleted fraction can be separated by 2 dimensional electrophoresis (2DE).
We are studying serum proteomics. This involves separation of serum proteins collected from cancer patients and healthy individuals, on 2D gels followed by visualization by silver staining. The presence of albumin and IgG hinders detection of low abundant proteins present in the serum; it also masks proteins which have same molecular weight and pI as albumin and IgG.
This problem was solved with the ProteoPrep® Blue Albumin and IgG Depletion Kit. Depleting albumin and IgG from serum before performing 2D separation results in better resolution and detection of low abundant proteins. This kit allows elution of proteins in tris-buffered urea solution, which can directly be used for 2DE; therefore, there is no need for precipitation of the depleted serum. I got better separation of serum proteins after using this kit without significant loss of other proteins.
I, therefore, recommend Sigma’s ProteoPrep® Blue Albumin and IgG Depletion Kit for quick and reproducible depletion of albumin and IgG and in order to get better resolution of low abundant proteins.
ProteoPrep® Blue Albumin and IgG Depletion Kit From Sigma-Aldrich
Depletion is rapid and reproducible and depleted extracts can be directly used for 2D applications.
It does not completely remove albumin and IgG.
The Bottom Line
It is quick, convenient, and reproducible way of specifically removing albumin and IgG from plasma/serum.