TY - JOUR
T1 - Cytosol estradiol receptor in human mammary carcinoma
T2 - An assay based on isoelectric focusing in polyacrylamide gel
AU - Wrange, Örian
AU - Nordenskjöld, Bo
AU - Gustafsson, Jan Åke
N1 - Funding Information:
The skillful and enthusiastic technical assistance of Miss Ingalill Ramberg and Mrs. Solveig Humla made this work possible. We also thank Dr. Anders Winter, LKB-Produkter AB, Bromma, Sweden, for fruitful discussions regarding technical problems of isoelectric focusing. This work was supported by grants from Cancerfiireningen i Stockholm, from Lotten Bohmans Fond, and from the Swedish Cancer Society.
Copyright:
Copyright 2018 Elsevier B.V., All rights reserved.
PY - 1978/4
Y1 - 1978/4
N2 - Limited trypsin digestion of the estradiol-receptor complex in human mammary cancer cytosol changed the sedimentation coefficient of the complex from 8 to 3-4 S. Gel filtration in high ionic strength on Sephadex columns calibrated with reference proteins showed that, after trypsinization, the Stokes radius of the estradiol-receptor complex decreased from 2.8 to 1.9 nm. Isoelectric focusing analysis indicated that the 8 S form of the steroid-receptor complex precipitated during electrofocusing in glass columns, whereas the trypsinized receptor focused as a sharp peak at pH 6.6. Based on these findings a method was developed for isoelectric focusing of the partially trypsin-digested estradiol-receptor complex in human mammary cancer cytosol on slabs of polyacrylamide gel. With this technique, the focusing time is 2 hr, at least eight different tumors may be analyzed on one gel, and sample volumes up to 300 μl are easily applied. Furthermore, electrofocusing of the estradiol-receptor complex was compared to sucrose gradient centrifugation analysis in low ionic strength and was found to be more sensitive and more specific. We therefore suggest that the described technique of isoelectric focusing should be considered as an alternative to sucrose gradient centrifugation for estradiol receptor assays.
AB - Limited trypsin digestion of the estradiol-receptor complex in human mammary cancer cytosol changed the sedimentation coefficient of the complex from 8 to 3-4 S. Gel filtration in high ionic strength on Sephadex columns calibrated with reference proteins showed that, after trypsinization, the Stokes radius of the estradiol-receptor complex decreased from 2.8 to 1.9 nm. Isoelectric focusing analysis indicated that the 8 S form of the steroid-receptor complex precipitated during electrofocusing in glass columns, whereas the trypsinized receptor focused as a sharp peak at pH 6.6. Based on these findings a method was developed for isoelectric focusing of the partially trypsin-digested estradiol-receptor complex in human mammary cancer cytosol on slabs of polyacrylamide gel. With this technique, the focusing time is 2 hr, at least eight different tumors may be analyzed on one gel, and sample volumes up to 300 μl are easily applied. Furthermore, electrofocusing of the estradiol-receptor complex was compared to sucrose gradient centrifugation analysis in low ionic strength and was found to be more sensitive and more specific. We therefore suggest that the described technique of isoelectric focusing should be considered as an alternative to sucrose gradient centrifugation for estradiol receptor assays.
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U2 - 10.1016/0003-2697(78)90243-9
DO - 10.1016/0003-2697(78)90243-9
M3 - Article
C2 - 565601
AN - SCOPUS:0018093689
VL - 85
SP - 461
EP - 475
JO - Analytical Biochemistry
JF - Analytical Biochemistry
SN - 0003-2697
IS - 2
ER -