Expression of genes in E. coli or yeast or baculovirus offers a convenient system to produce large amounts of recombinant proteins that may otherwise be difficult to isolate from natural cells and tissues. Very often antibodies to these newly identified proteins are not available to study its biochemical properties, monitor protein expression, and purification. In order to circumvent this problem, short pieces of well-defined peptides (Poly-His, Flag-epitope or c-myc epitope or HA-tag) or small proteins (bacterial GST, MBP, Thioredoxin, b-Galactosidase, VSV-Glycoprotein, etc.) are often cloned along with the target gene. Proteins are expressed as fusion proteins. Antibodies to these fusion-tags are already available to monitor fusion protein expression and purification. Therefore, fusion-tags serve as universal tags much like secondary antibodies. Many tags have their own characteristics. Poly-His-fusion proteins (6 x His) can bind to Nickel-Sepharose or Nickel-HRP. GST-fusion proteins can bind to glutathione-Sepharose. Therefore, a high degree of purification of fusion protein can be achieved in just one affinity purification step. Purity of fusion proteins can be followed by Tag-antibodies. Very often, fusion proteins are directly injected into animals to generate antibodies. Some fusion tags can be removed later by treatment with enzymes to generate tag-free recombinant proteins.
Source:Bacterial GST (Schistosoma japonicum, ~27kD) expressed in E. coli. Purified GST was coupled to agarose at ~5mg/ml of beads. The column has a binding capacity of ~2-5mg anti-GST/ml of beads.
Applications:The GST-agarose column can be used to remove the anti-GST antibodies. Other applications not tested.
Recommended Dilution:We recommend processing ~1ml antiserum per 0.25ml of the beads or it can be scaled up accordingly. Load antiserum diluted 1:5 in PBS to adsorb anti-GST at room temp. Collect unbound fraction containing GST-depleted antiserum. It may be necessary to repeat this adsorption if the sample contain high concentrations of ant-GST. Optimal dilutions to be determined by the researcher.
Column Regeneration:The column can be regenerated by passing 3ml of 0.1M Glycine buffer, pH 2.5, and then immediately washing with PBS, pH 7.4 with 10-20 volumes.
Storage and Stability:May be stored at 4°C before opening. DO NOT FREEZE! Stable at 4°C as an undiluted liquid. Dilute only prior to immediate use. Stable for at least 6 months.
