Isolation and characterization of murine cell surface components: I. Purification of milligram quantities of thy-1.1*

Academic Article

Abstract

  • The Thy-l.1 molecule was isolated from the BW5147 murine lymphoblastoid cell line. The initial step in purification was the preparation of a crude plasma membrane fraction followed by acetone precipitation. The acetone pellet was solubilized using deoxycholate (DOC) and Thy-1.1 was purified by use of a Lens culinaris lectin affinity column and an AcA-34 gel filtration column. The purified glycoprotein with Thy-1.1 activity had a mol wt of approximately 25,000 daltons. The isolation of this molecule was effected by detecting Thy-I activity utilizing rabbit anti-mouse brain serum tested on rat thymocytes. Congenic anti-Thy-l.1 serum was ineffective in detecting Thy-l.1 after DOC solubilization. An antiserum prepared in rabbits to the purified Thy-1.1 was found to be cytotoxic to mouse and rat thymocytes. The cytotoxic activity of this antisera could be completely absorbed with AKR/Jax brain and thymus but was not absorbed by liver. In addition, AKR/Jax thymocytes totally absorbed all cytotoxic activity of the rabbit anti-purified Thy-1 serum for BW5147 cells suggesting that the cell line shares identical specificities with normal thymocytes. The purified Thy-1.1 molecule was able to totally absorb the cytotoxic activity of mouse congenic anti-Thy-1. These studies serve as a model for the isolation of other murine lymphoid cell surface components in quantities for detailed structural and functional analysis. © American Society for Clinical Pathology.
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    Digital Object Identifier (doi)

    Author List

  • Zwerner RK; Barstad PA; Acton RT
  • Start Page

  • 986
  • End Page

  • 1000
  • Volume

  • 146
  • Issue

  • 4