Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis.
Antibody cross-linking and target elution protocols used for immunoprecipitation significantly modulate signal-to noise ratio in downstream 2D-PAGE analysis.
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DOI：
10.1186/1477-5956-9-45
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发表时间：
2011-08-04
期刊：
影响因子：
2
通讯作者：
Slupphaug G
中科院分区：
文献类型：
--
作者：
Sousa MM;Steen KW;Hagen L;Slupphaug G
免疫沉淀和随后的2D-PAGE/质谱是研究翻译后蛋白质修饰的有力工具。在该工作流程中经常忽略的是化学交联剂对抗体亲和力的影响，以及在2D-PAGE中常用的缓冲液中主要靶蛋白的不完全洗脱。这可能会妨碍非丰富蛋白质亚型的检测。在此，我们比较了使用SDS或BS 3将抗体与Dynabeads®蛋白A交联，以及在2D-PAGE分离前各种靶洗脱缓冲液的效率。BS 3交联通常导致的非特异性结合比BS 4少，而BS 4交联产生的靶蛋白的总产量更高。无论使用的交联剂如何，用常规的基于甘氨酸或尿素的缓冲液观察到靶蛋白的不完全洗脱。相反，用2%热SDS获得完全洗脱，随后在含有4%CHAPS的尿素缓冲液中稀释至0.2%最终SDS，得到适合于质谱分析的完全聚焦的凝胶。IG交联剂的选择以及在下游2D-PAGE之前在SDS中有效洗脱目的蛋白可能是分析磁珠免疫沉淀富集的低丰度蛋白的关键因素。
Immunoprecipitation and subsequent 2D-PAGE/mass spectrometry are powerful tools to study post-translational protein modifications. Often disregarded in this workflow is the impact of the chemical cross-linker upon antibody affinity, as well as incomplete elution of primary target protein in buffers commonly used in 2D-PAGE. This may impede detection of non-abundant protein isoforms. Here we have compared cross-linking of antibodies to Dynabeads® Protein A by using DMP or BS3, as well as the efficiency of various target elution buffers prior to 2D-PAGE separation. BS3 cross-linking generally resulted in less non-specific binding than DMP, whereas DMP cross-linking gave overall higher yield of target protein. Regardless of the cross-linker used, incomplete elution of target protein was observed with conventional glycine- or urea-based buffers. Conversely, complete elution was obtained with 2% hot SDS and subsequent dilution in urea buffer containing 4% CHAPS, to 0.2% final SDS yielded perfectly focused gels suitable for mass spectrometry analysis. Careful choice of Ig cross-linker as well as efficient elution of target protein in SDS prior to downstream 2D-PAGE may be key factors to analyze low-abundance proteins enriched by magnetic bead immunoprecipitation.
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影响因子：
2
作者：
Chevalier F
通讯作者：
Chevalier F
影响因子：
2.9
作者：
Zuobi-Hasona, K;Crowley, PJ;Brady, LJ
通讯作者：
Brady, LJ
影响因子：
2.2
作者：
GERSTEN, DM;MARCHALONIS, JJ
通讯作者：
MARCHALONIS, JJ
影响因子：
7.4
作者：
Shevchenko, A;Wilm, M;Mann, M
通讯作者：
Mann, M
DOI：
10.1038/sj.emboj.7601958
发表时间：
2008-01-09
期刊：
The EMBO journal
影响因子：
--
作者：
Hagen L;Kavli B;Sousa MM;Torseth K;Liabakk NB;Sundheim O;Pena-Diaz J;Otterlei M;Hørning O;Jensen ON;Krokan HE;Slupphaug G
通讯作者：
Slupphaug G