Immunoprecipitation – Role of Glutathione Sepharose 4B And Protein A Sepharose
Glutathione (GSH) is an antioxidant produced by the body to combat aging and sickness. Well, the good news is that glutathione is produced by the human body. However, glutathione production and, consequently, its levels in the body are depleted due to a number of factor s: stress, pollution, poor diet,aging, infections, medications, etc., being some of them.
Reduced glutathione levels mean unrestrained cell damage due to oxidative stress. Detoxification is less and you become more prone to diseases and infections.
Glutathione is produced in the body naturally all the time and is not an essential nutrient. It is a combination of the amino acid building blocks of cysteine, glycine and glutamine. All cells in the human body are capable of synthesizing glutathione. However, studies have shown that glutathione synthesis by the liver cells is crucial and absolutely essential.
Glutathione can also exist in the oxidized form of glutathione disulphide (GSSG) in the cells. Healthy cells have more than 90% of glutathione in the reduced form and the remaining 10% in the oxidized form. The increased ratio of the oxidized to the reduced forms is used as an indicator of oxidative stress.
The main functions of glutathione are as follows:
• It helps to recharge other antioxidants such as vitamins C and E in the cells and maintain them in their reduced forms.
• It is critical for proper immune function in the body.
• It acts as a detoxifier; toxins and free radicals, and molecules of heavy metals stick to the sulfur chemical groups that glutathione contains.
• It aids crucial activities in the body such as DNA synthesis, enzyme activation, protein synthesis, iron metabolism, nitric oxide regulation, etc.
Supplementation to maintain healthy levels of glutathione is therefore very important. Studies have indicated that direct a glutathione supplement is ineffective and not absorbed properly in the gastrointestinal tract. Until the discovery byDr. Robert Keller that resulted in the supplement MaxGXL.
Glutathione’s Role in Immunoprecipitation
The technique of immunoprecipitation is all about using the scientific fact that by using an antibody that specifically binds to a protein can be used to precipitate and further isolate the particular protein from a mixture that contains thousands of different proteins.
The generation of an antibody that binds to a single targeted known protein has always been difficult. This is overcome when tags are inserted to the C- or N- end of the specific protein.
This process offers the advantage that the same tags can be reused on various proteins and the same antibody can also be repeatedly used. One such tag used is Glutathione-S-transferase (GST).
Direct immunoprecipitation is possible by immobilizing the antibodies for a specific protein on a solid-phase substrate such as microscopic agarose/sepharose beads or resins/slurries. The sponge-like structure of the sepharose particle is available for binding the antibodies. These antibodies, in turn, will bind the target protein. The advantage offered is the high binding capacity without the need to use specialized equipment. However, to realize the full advantage of the method, the amount of antibodies used in the process of immunoprecipitation is crucial.
Insufficient amounts result in incomplete coating of the sepharose with antibodies. The remaining portions are open for any other non-specific binding. This makes effective data interpretation difficult. Protein A- sepharose purportedly has specific advantages over protein A- agarose when immunoprecipitating a protein from a cell lysate. A sepharose is claimed to be a sufficiently aged agarose. However, sepharose is more stable than agarose.
Glutathione Sepharose 4b
Glutathione sepharose 4B is a product of GE Healthcare. It is used for the purification of GST-tagged proteins from lysates or other proteins that are glutathione dependent. Glutathione sepharose 4B contains a coupling that is optimized to give high binding capacities for GST-tagged proteins. The factors that determine the dynamic binding capacity include target protein, flow rate, pH, temperature, etc., among many others. Lower flow rates improve the binding capacity. Size and concentration of the protein loaded also determine the binding of the GST-tagged proteins.
The Process of Purification
• The actual purification process involves the preparation of the buffer solution of high purity and preparation of the sample. Care should be taken to see that the sample is not too viscous and it is a good idea to filter the sample before the batch purification process.
• The next step is to prepare the glutathione sepharose 4B; it is delivered in ethanol. However, a 50% slurry is used for the purification. The slurry is washed by addition of the binding buffer repeatedly, centrifugation and further decantation.
• Thereafter the cell lysate is added to the glutathione sepharose 4B, incubated for period of 30 minutes at room temperature and then agitated. Transfer the mixture to a tube and repeat a sedimentation of the medium, decantation of the flow-through,and washing with a binding buffer three times.
• An elution buffer is then added, incubated at room temperature for 5 to 10 minutes, and the resulting mixture thereafter agitated end-to-end. This helps to elute the protein. This is followed by sedimentation and decantation and the whole process is repeated thrice. The purity of the protein that is eluted is determined thrice.
Kinetics of GST
Sufficient time must be allowed to enable the maximum binding capacity. The kinetics of GST binding with glutathione is relatively slow. The binding efficiency varies between the different GST-tagged proteins. Volumes used and time required for individual processes may also differ. Glutathione sepharose can be reused for other samples. However, it depends on the nature of the sample. Sufficient care should be taken to avoid cross contamination.
Protein G and protein A have been used in several matrices and provide a very effective method of separating IgG and other subclasses of IgG from cell-culture flow-throughs and serum. IgG are antibody molecules that bind to pathogens like virus bacteria and fungi and neutralize their toxins. Protein A and protein G are bacterial proteins from Group G Streptococci and Staphylococcus aureus bacteria. Protein G and A have a high affinity for the Fc region of IgGof many species.
When combined with Glutathione sepharose base they form useful media that can be used to purify antibodies. GE Healthcare offers both native and recombinant versions of Protein A coupled to sepharose that help in immunoglobulin purification and fractionation. Many studies have indicated rapid and efficient immunoglobulin yield when fractionated with protein A sepharose.
