Capturing sodium dodecyl sulfate-treated protein antigens by antibody affinity electrophoresis.
Modifications to antibody affinity electrophoresis for improved detection of proteins have been developed. The bifunctional linker glutaraldehyde is added to the polyacrylamide gel solution for better incorporation of the bait antibody into a distinct region of a 10% w/v polyacrylamide gel. The addition of glutaraldehyde alleviates the need of an electrophoresis buffer with a specific pH. The protein sample to be analyzed is treated with 2% w/v sodium dodecyl sulfate (SDS) to ensure that they carry a negative charge. The negative charge will allow the proteins to migrate towards the cathode and hence pass through the area embedded with the bait antibody. It is observed that electrophoretic migration of bovine serum albumin (BSA) or protein G ceases upon encounter with anti-BSA whereas proteins ovalbumin, beta-lactoglobulin A, and myoglobin migrate freely. However, the addition of 0.1% w/v SDS in the native gel running buffer disrupts the antibody-antigen bond and neither BSA nor protein G can be captured by anti-BSA.
Lee
Bao-Shiang
BS
Protein Research Laboratory, Research Resources Center, University of Illinois, Chicago, IL 60612, USA. boblee@uic.edu
Krishnanchettiar
Sangeeth
S
Lateef
Syed Salman
SS
Gupta
Shalini
S
D000906:Antibodies / Q000737:chemistry; D000937:Antigen-Antibody Reactions; D000941:Antigens / Q000737:chemistry / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005976:Glutaral / Q000737:chemistry; D007074:Immunoglobulin G / Q000737:chemistry; D009419:Nerve Tissue Proteins / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012710:Serum Albumin, Bovine / Q000737:chemistry / Q000302:isolation & purification; D012967:Sodium Dodecyl Sulfate / Q000737:chemistry
D000906:Antibodies; D000941:Antigens; C029152:G-substrate; D007074:Immunoglobulin G; D009419:Nerve Tissue Proteins; D011506:Proteins; D012710:Serum Albumin, Bovine; D012967:Sodium Dodecyl Sulfate; D005976:Glutaral