eCite Digital Repository

Elution of snapper, Pagrus auratus (Bloch and Schneider) Ig from a protein A affinity chromatography column yields contamination from the binding ligand

Citation

Morrison, RN and Nowak, BF, Elution of snapper, Pagrus auratus (Bloch and Schneider) Ig from a protein A affinity chromatography column yields contamination from the binding ligand, Bulletin of European Association of Fish Pathologists, 20, (5) pp. 174-179. ISSN 0108-0288 (2000) [Refereed Article]

Abstract

Staphylococcal protein A (SpA) affinity chromatography was used to single step purify immunoglobulins (Ig) from snapper (Pagrus auratus) serum. Elution of Ig was successful, with analysis of the eluent using SDS-PAGE under fully reducing conditions and PAGE under native conditions revealing that the product was of high purity. Polyclonal antisera to SpA purified snapper Ig was produced in rabbits. Probing of reduced purified Ig with the rabbit-anti-snapper Ig antisera in a Western blot demonstrated that the antisera were most reactive with the heavy (H) chains but also with an unknown protein of approximately 65 kDa in molecular weight. Non-immune rabbit serum (pre-bleed) was not reactive with the H chains of the reduced Ig however, reaction with the unknown protein (65 kDa) occurred. Further, probing of purified Ig in a Western blot with a suite of control sera and antisera demonstrated that the unknown protein was reactive with all mammalian sera and antisera tested. SpA affinity chromatography without prior application of snapper serum was performed. Eluent was probed in a Western blot by rabbit-anti-snapper Ig antisera and non-immune rabbit sera (pre-bleed) once more and were also reactive. In addition, elution buffer pH (3-5) had no effect on the elution of the unknown protein. In contrast, only H and light (L) chains of reduced snapper serum were positive when probed with the antisera. It was concluded that due to the non-specific binding in the Western blot the unknown protein was SpA contamination.

Item Details

Item Type:Refereed Article
Research Division:Agricultural and Veterinary Sciences
Research Group:Fisheries Sciences
Research Field:Fish Pests and Diseases
Objective Division:Animal Production and Animal Primary Products
Objective Group:Fisheries - Aquaculture
Objective Field:Fisheries - Aquaculture not elsewhere classified
Author:Morrison, RN (Dr Richard Morrison)
Author:Nowak, BF (Professor Barbara Nowak)
ID Code:18516
Year Published:2000
Web of Science® Times Cited:4
Deposited By:TAFI - Aquaculture
Deposited On:2000-08-01
Last Modified:2001-03-20
Downloads:0

Repository Staff Only: item control page