Multiple ERbeta antisera label in ERbeta knockout and null mouse tissues.
In the process of characterizing a custom-made affinity-purified antiserum for estrogen receptor beta (ERbeta), ck5912, we used a number of common tests for specificity of ck5912 along with that of 8 commercially available ERbeta antisera: Affinity Bioreagents PA1-310B, Invitrogen D7N, Upstate 06-629, Santa Cruz H150, Y19, L20, 1531, and Abcam 9.88. We tested their recognition of recombinant ERbeta (rERbeta) versus rERalpha, ERbeta versus ERalpha transfected into cell lines, as well as labeling in wildtype (WT) versus estrogen receptor beta knockout (betaERKO) and null (ERbeta(ST)(L-/L-)) mouse ovary, hypothalamus, and hippocampus. To our surprise, we found that while most of these antisera passed some tests, giving the initial impression of specificity, western blot analysis showed that all of them recognized apparently identical protein bands in WT, betaERKO and ERbeta(ST)(L-/L-) tissues. We share these results with the goal of helping other researchers avoid pitfalls in interpretation that could come from use of these ERbeta antisera.
Snyder
Melissa A
MA
Department of Neurobiology and Physiology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA. m-ramous@northwestern.edu
Smejkalova
Tereza
T
Forlano
Paul M
PM
Woolley
Catherine S
CS
D000818:Animals; D000906:Antibodies / Q000032:analysis / Q000276:immunology* / Q000302:isolation & purification*; D000918:Antibody Specificity / Q000276:immunology*; D015153:Blotting, Western; D001921:Brain / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D002460:Cell Line; D047629:Estrogen Receptor beta / Q000235:genetics / Q000276:immunology*; D005260:Female; D018390:Gene Targeting; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D010053:Ovary / Q000166:cytology / Q000276:immunology / Q000378:metabolism; D011993:Recombinant Fusion Proteins / Q000276:immunology; D014162:Transfection
D000906:Antibodies; D047629:Estrogen Receptor beta; D011993:Recombinant Fusion Proteins