Pierce’s CarboLink Coupling Gel is an affinity support for immobilizing glycoproteins through oxidized sugar groups. It is ideal for immobilizing polyclonal antibodies, which contain abundant carbohydrates located on the Fc portion of the molecule. Because such antibodies are coupled to the CarboLink Gel through the Fc portion only, they are properly oriented with their antigen-binding sites unobstructed, offering greater purification capability. Some monoclonal antibodies may be immobilized using CarboLink Gel, provided they contain an adequate amount of carbohydrate.
The immobilization chemistry uses sodium meta-periodate to oxidize cis -glycol groups in sugars of polysaccharide moieties. The resulting aldehydes then react spontaneously with hydrazide groups on the CarboLink Gel to form stable hydrazone bonds. The coupling conditions are flexible with regard to time and temperature. The long spacer arm reduces steric hindrance and the support has minimal nonspecific binding characteristics, making this an excellent gel for affinity chromatography. When coupled to stable glycoproteins, the gel support may be regenerated and reused at least 10 times. There are various crosslinking agents, which can be used to crosslink antibodies, such as disuccinimidyl suberate (DSS), disuccinimidyl glutarate (DSG), Bis[sulfosuccinimidyl] suberate (BS3), dimethyl adipimidate (DMA), dimethyl suberimidate (DMS) and dimethyl pimelimidate (DMP), but all of them react with the primary amine group of the amino acids in the antibodies which might cause the antigen binding capacity of these antibodies to be obstructed due to steric hindrance.
CarboLink Coupling Gel comes in a pack of 10 ml as 50% slurry, which is enough to couple 5 to 100 mg of glycoprotein. The other reagents required for the coupling procedure are sodium meta-periodate and a desalting column or 30,000 cutoff filters; the remaining reagents are routinely used chemicals. 10-25 mM of sodium meta-periodate is dissolved in 1 ml glycoprotein solution (0.5-10 mg antibody in 0.1M phosphate buffer, pH 7) in an amber vial and kept for 30 min at room temperature. The excess of periodate is then removed by passing oxidized glycoprotein solution through the 30,000 cutoff filters with an excess of 0.1 M phosphate buffer. Oxidized glycoprotein is recovered in 1 ml of 0.1 M phosphate buffer. Then 2 ml of coupling gel is placed in a 2 ml tube and kept at 4ºC for 15 min to allow the gel beads to settle. The supernatant is then discarded and the beads are washed with 0.1 M phosphate buffer 3 times. During the last wash, after removing the supernatant, 1 ml of oxidized glycoprotein is added to these gel beads and kept at 4ºC on a rocker, overnight. After incubation, the tube is kept upright for 15 min to allow the gel to settle and then the supernatant is removed and the gel beads are washed 3 times with 0.1 M phosphate buffer. The supernatant can be collected and the absorbance can be taken to check the coupling efficiency. The column can be stored by washing with 10 gel bed volumes of 1 M NaCl, then with 5 gel bed volumes of PBS and finally, storing in 2 ml PBS at 4ºC.
Affinity purification can be done by incubating 1.5 ml of the protein sample (in PBS) with the column on rocker for 1 h at room temperature. The column is then spun at 7000 rpm for 10 sec and the supernatant is removed; this is followed by 3 washes with 3 ml PBS. The protein is eluted 4 or more times in a buffer comprised of 450 ul of 0.1 M glycine buffer (pH-2.7) and 50 ul of Tris-Cl (pH 8.8). Fractions can be monitored by taking the absorbance at 280 nm and fractions of interest are pooled, ethanol precipitated and dissolved in PBS. The column is washed with 5 ml of PBS and stored in 2 ml of PBS with 0.05% sodium azide.
My research aims to identify proteins/tumor antigens which elicit an antibody response in cancer. I perform Protein A-Sepharose based immunoprecipitation of tumor cell proteins with IgGs purified from cancer patients and from healthy individuals. In order to detect proteins which elicit an antibody response in cancer patients, the immunoprecipitate is then run by SDS-PAGE and proteins are transferred to PVDF membrane and immunostained with the corresponding IgGs. The blot is detected by chemiluminescent detection. The resulting film image shows reactive bands which represent tumor antigens, but it also gives background staining because of IgG contamination. This makes detection of proteins with a molecular weight close to IgG heavy and light chains difficult.
Immobilizing IgGs using CarboLink Coupling Gel solved this problem. As eluted proteins after immunoprecipitation are free of IgG contamination, proteins having molecular weight close to IgGs could be detected with out any problem. In addition, since the immobilization reaction occurs though Fc portion only, the antigen binding capacity of antibodies remained unobstructed.
I would, therefore, recommend CarboLink Coupling Gel for coupling polyclonal antibodies, which can be used in affinity purification.
Sanjeev Shukla
PhD Student
Advanced Centre for Treatment, Research and Education in Cancer (ACTREC)
Biochemistry and Cell Biology Dept