B-PER GST Fusion Protein Spin Purification Kit From Thermo Scientific Pierce Protein Research Products

B-PER GST Fusion Protein Spin Purification Kit provides rapid purification of glutathione S-transferase (GST) fusion proteins from bacteria. GST fusion proteins can be purified using glutathione sepharose (available commercially) with the help of additional manually made reagents, but doing so generally requires the use of sonication during lysis of bacterial cells, which may disrupt the integrity of GST-fusion proteins; it also takes more time. Although different reagents are available that do not require sonication for bacterial lysis, Pierce’s kit provides a simple, efficient solution for lysis followed by glutathione agarose based rapid purification of GST fusion proteins using spin columns.

The kit contains reagents sufficient for 16 GST fusion protein purifications, each from a 250 ml bacterial culture. This kit contains 165 ml of B-PER bacterial protein extraction reagent, 16 ml of immobilized glutathione slurry, 85 ml of wash buffer, 16 x 15 mg reduced glutathione, 16 spin columns, and 80 collection tubes. Other reagents required are protease inhibitors.

The purification protocol is simple and quick. 250 ml of bacterial culture with an OD₆₀₀ of 1.5 – 3 is pelleted down by centrifugation at 5000 rpm for 10 mins at 4ºC. Pelleted cells are suspended in 10 ml of B-PER reagent by pipetting up and down and then mixing on a rocker for 10 mins at room temperature (RT). At this point, protease inhibitors can be added. Now the bacterial cell lysate is separated from the insoluble material by centrifugation at 14000 rpm at 4ºC for 10 mins. The supernatant is taken in a 15 ml conical tube and 1 ml of glutathione agarose slurry is added with a wide-bore tip after swirling glutathione agarose slurry to obtain homogenous slurry. The tube is incubated on rocker at RT for 10 mins. Now it is centrifuged at 1000 rpm at 4ºC for 5 mins. The supernatant is discarded and the pellet is resuspended in 250 ul of wash buffer which brings the total volume to 750 ul. This gel slurry is transferred to the spin column with collection tube. The assembly is centrifuged at 1000 rpm for 2 mins at 4ºC and the spin column is transferred to a new collection tube. Now the elution buffer is prepared by adding 2 ml of the wash buffer to one of the 16 vials containing 15 mg of reduced glutathione. To the spin column, 500 ul of the fresh reduced glutathione is added and incubated for 5 mins and the GST fused protein is eluted by centrifuging at 1000 rpm for 2 mins at 4ºC. This elution is repeated 3 more times. The eluted protein can be used directly in downstream applications.

My research aims to identify tumor antigens, proteins which elicit antibody response in cancer. It involves screening recombinant tumor antigens with serum IgG from cancer patients. In order to purify recombinant proteins, they are GST tagged. The GST fusion recombinant proteins can be easily purified using glutathione sepharose, reduced glutathione and other routinely used lab chemicals, but the method is time consuming and some degradation of the protein also occurs because of the ultrasonication process required to lyse the cells. The B-PER GST Fusion Protein Spin Purification Kit provides an easy and quick way of purifying GST fusion proteins. This method doesn’t include an ultrasonication step in the protocol and thus, the protein can be purified in short time, in tact, with ease.

I recommend the B-PER B-Per GST Fusion Protein Spin Purification Kit for quick and easy purification of GST fusion proteins from bacterial systems.