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使用地高辛配体系统消除非放射性电泳迁移率变动分析中的非特异性条带。

Elimination of nonspecific bands in non-radioactive electrophoretic mobility shift assays using the digoxigenin system.

作者信息

Tokunaga Saimi, Stegeman John J

机构信息

Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.

Biology Department, Woods Hole Oceanographic Institution, Woods Hole, MA 02543, USA.

出版信息

Anal Biochem. 2014 Nov 15;465:70-2. doi: 10.1016/j.ab.2014.06.020. Epub 2014 Jul 5.

DOI:10.1016/j.ab.2014.06.020
PMID:25004462
原文链接:https://pmc.ncbi.nlm.nih.gov/articles/PMC4318779/
Abstract

In the course of detecting nuclear transcription factors by electrophoretic mobility shift assay using digoxigenin (DIG)-labeled probes, we encountered a problem with a considerable nonspecific shift band in negative control lanes from which protein extracts were omitted. This nonspecific shift band can interfere with the detection of the desired target protein. Purification of the DIG-labeled probes by removing unincorporated DIG-labeled nucleotides did not resolve the problem. However, the introduction of an additional step of heating at 95 °C for 5 min and subsequent reannealing after DIG-labeled probe synthesis eliminated these nonspecific shift bands and allowed accurate analysis of the target protein.

摘要

在用地高辛（DIG）标记的探针通过电泳迁移率变动分析检测核转录因子的过程中，我们在未加入蛋白质提取物的阴性对照泳道中遇到了一个相当明显的非特异性条带迁移问题。这种非特异性条带迁移会干扰所需靶蛋白的检测。通过去除未掺入的DIG标记核苷酸来纯化DIG标记的探针并不能解决这个问题。然而，在DIG标记探针合成后引入额外的95℃加热5分钟及随后重新退火的步骤消除了这些非特异性条带迁移，并允许对靶蛋白进行准确分析。

https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ec2/4318779/bbf692ca584a/nihms627051f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ec2/4318779/397d18ed2ed1/nihms627051f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ec2/4318779/bbf692ca584a/nihms627051f2.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ec2/4318779/397d18ed2ed1/nihms627051f1.jpg
https://cdn.ncbi.nlm.nih.gov/pmc/blobs/5ec2/4318779/bbf692ca584a/nihms627051f2.jpg

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