Purification of murine immunoglobulin M from spent tissue culture supernatant by one-step gel filtration chromatography procedure.
Optimal purification of immunoglobulin M (IgM) grown as spent tissue culture supernatant can be achieved by a straightforward size-exclusion chromatography procedure. Preparative isolation of IgM was achieved by precipitation with saturated ammonium sulphate to achieve a 45% solution. IgM was then purified by gel filtration chromatography with a solution of 100 mM Tris + 150 mM NaCl at pH 8. Denaturing electrophoresis and Western immunoblotting revealed two prominent bands at 80 and 25 kDa, indicative of the heavy and light chains of IgM respectively. The specific immunoreactivity of this IgM was assessed by a modified enzyme antigen capture assay. In contrast to affinity and ion-exchange chromatographies, gel filtration allows retention of IgM immunoreactivity.
Novales-Li
P
P
Department of Pharmacology, University of Oxford, UK.
D000110:Acetylcholinesterase / Q000276:immunology; D000818:Animals; D015153:Blotting, Western; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D007075:Immunoglobulin M / Q000302:isolation & purification*; D051379:Mice; D011489:Protein Denaturation
D007075:Immunoglobulin M; D000110:Acetylcholinesterase