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 language | English |
|---|---|
| Pages (from-to) | 315-319 |
| Number of pages | 5 |
| Journal | Immunology Letters |
| Volume | 7 |
| Issue number | 6 |
| DOIs | |
| Publication status | Published - 1984 |
| Externally published | Yes |
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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 journal › Article
}
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 -