Department of Neurobiology and Physiology, Northwestern University, 2205 Tech Drive, Evanston, IL 60208, USA.
J Neurosci Methods. 2010 May 15;188(2):226-34. doi: 10.1016/j.jneumeth.2010.02.012. Epub 2010 Feb 16.
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.
在对一种定制的雌激素受体β（ERβ）亲和纯化抗血清 ck5912 的特性进行描述的过程中，我们使用了一些常见的特异性测试方法来评估 ck5912，同时还评估了 8 种市售的 ERβ抗血清：Affinity Bioreagents PA1-310B、Invitrogen D7N、Upstate 06-629、Santa Cruz H150、Y19、L20、1531 和 Abcam 9.88。我们测试了它们对重组 ERβ（rERβ）与 rERα、转染到细胞系中的 ERβ与 ERα的识别能力，以及在野生型（WT）与雌激素受体β敲除（βERKO）和缺失（ERβ（ST）（L-/L-））小鼠卵巢、下丘脑和海马中的标记。令我们惊讶的是，虽然这些抗血清中的大多数通过了一些测试，给人以特异性的初步印象，但 Western blot 分析显示，它们都能识别 WT、βERKO 和 ERβ（ST）（L-/L-）组织中明显相同的蛋白条带。我们分享这些结果的目的是帮助其他研究人员避免因使用这些 ERβ抗血清而导致的解释上的陷阱。
