Purification of mRNA for immunoglobulin κ-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA

S. M. Deyev, R. S. Mukhamedov, N. K. Sakharova, O. L. Polyanovsky, V. Viklický, F. Franěk, J. Hradec

Research output: Contribution to journalArticle

Abstract

The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin κ-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3′-untranslated region and a part of the constant region of the κ-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52°C and by elution at 60 °C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the κ-chain, was identified as the only translation product.

Original languageEnglish
Pages (from-to)315-319
Number of pages5
JournalImmunology Letters
Volume7
Issue number6
DOIs
Publication statusPublished - 1984
Externally publishedYes

Fingerprint

Immunoglobulin Subunits
Immobilized Nucleic Acids
Hybridomas
Complementary DNA
Messenger RNA
Cell-Free System
3' Untranslated Regions
Radioactivity
Immunoglobulins
Swine
Peptides
Antibodies
DNA
Genes

Keywords

  • diazobenzyloxymethyl- cellulose
  • immunoprecipitation
  • polyacrylamide gel electrophoresis
  • RNA-DNA hybridization
  • translation in cell- free system

ASJC Scopus subject areas

  • Immunology
  • Immunology and Allergy

Cite this

Purification of mRNA for immunoglobulin κ-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA. / Deyev, S. M.; Mukhamedov, R. S.; Sakharova, N. K.; Polyanovsky, O. L.; Viklický, V.; Franěk, F.; Hradec, J.

In: Immunology Letters, Vol. 7, No. 6, 1984, p. 315-319.

Research output: Contribution to journalArticle

Deyev, S. M. ; Mukhamedov, R. S. ; Sakharova, N. K. ; Polyanovsky, O. L. ; Viklický, V. ; Franěk, F. ; Hradec, J. / Purification of mRNA for immunoglobulin κ-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA. In: Immunology Letters. 1984 ; Vol. 7, No. 6. pp. 315-319.
@article{c76ba765966549a48e8914df9ccfe837,
title = "Purification of mRNA for immunoglobulin κ-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA",
abstract = "The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin κ-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3′-untranslated region and a part of the constant region of the κ-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52°C and by elution at 60 °C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the κ-chain, was identified as the only translation product.",
keywords = "diazobenzyloxymethyl- cellulose, immunoprecipitation, polyacrylamide gel electrophoresis, RNA-DNA hybridization, translation in cell- free system",
author = "Deyev, {S. M.} and Mukhamedov, {R. S.} and Sakharova, {N. K.} and Polyanovsky, {O. L.} and V. Viklick{\'y} and F. Franěk and J. Hradec",
year = "1984",
doi = "10.1016/0165-2478(84)90087-7",
language = "English",
volume = "7",
pages = "315--319",
journal = "Immunology Letters",
issn = "0165-2478",
publisher = "Elsevier",
number = "6",

}

TY - JOUR

T1 - Purification of mRNA for immunoglobulin κ-chains from myeloma and hybridoma cells using hybridization to immobilized complementary DNA

AU - Deyev, S. M.

AU - Mukhamedov, R. S.

AU - Sakharova, N. K.

AU - Polyanovsky, O. L.

AU - Viklický, V.

AU - Franěk, F.

AU - Hradec, J.

PY - 1984

Y1 - 1984

N2 - The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin κ-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3′-untranslated region and a part of the constant region of the κ-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52°C and by elution at 60 °C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the κ-chain, was identified as the only translation product.

AB - The principle of mRNA purification by hybridization to an immobilized DNA fragment was applied to the isolation of mRNA coding for immunoglobulin κ-chains of mouse myeloma MOPC 21 and mouse hybridoma PTF-02. The DNA fragment comprising the 3′-untranslated region and a part of the constant region of the κ-chain gene was covalently attached to diazobenzyloxymethyl-cellulose and used as an affinity adsorbent. A homogeneous 14S mRNA species was obtained by hybridization of total mRNA to the affinity adsorbent at 52°C and by elution at 60 °C. Addition of the purified mRNA to a fractionated cell-free translation system resulted in a significant increase in the radioactivity immunoprecipitated by pig anti-mouse immunoglobulin antibodies. A single radioactive polypeptide of apparent Mr of 25,000, corresponding obviously to the κ-chain, was identified as the only translation product.

KW - diazobenzyloxymethyl- cellulose

KW - immunoprecipitation

KW - polyacrylamide gel electrophoresis

KW - RNA-DNA hybridization

KW - translation in cell- free system

UR - http://www.scopus.com/inward/record.url?scp=0021251108&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=0021251108&partnerID=8YFLogxK

U2 - 10.1016/0165-2478(84)90087-7

DO - 10.1016/0165-2478(84)90087-7

M3 - Article

AN - SCOPUS:0021251108

VL - 7

SP - 315

EP - 319

JO - Immunology Letters

JF - Immunology Letters

SN - 0165-2478

IS - 6

ER -