MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.
MILKSHAKE: novel validation method for antibodies to post-translationally modified targets by surrogate Western blot.
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DOI：
10.2144/btn-2021-0078
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发表时间：
2022-01
期刊：
影响因子：
2.7
通讯作者：
中科院分区：
文献类型：
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抗体（Ab）验证是指在给定应用中彻底测定Ab的灵敏度和特异性的程序。针对免疫后修饰（PTM）靶标的Ab的验证特别具有挑战性，因为它需要专门制备的抗原。在这里，我们描述了一种新的验证方法，在Western印迹中使用替代蛋白。我们称之为“MILKSHAKE”的替代蛋白是一种修饰的麦芽糖结合蛋白，其酶促缀合至来自所选靶标的肽，所述肽在感兴趣的残基处修饰或未修饰。残基修饰状态的确定性可用于确认Ab特异性。该方法还允许即使在不存在经处理的细胞裂解物或经处理的细胞裂解物的可用性有限的情况下进行Ab验证。我们已经开发了一种新的方法，用于验证抗体在Western印迹使用修饰的麦芽糖结合蛋白酶促缀合到与哺乳动物细胞裂解物混合的靶肽作为抗原。该方法可用于确认一抗对靶序列的特异性，并作为对所用一抗特异性的Western印迹过程对照。我们将证明MILKSHAKE用于后修饰（PTM）靶点和用于确定氨基酸多态性位点的特异性的实用性，但很明显，它也可以用于非PTM。
Antibody (Ab) validation is the procedure in which an Ab is thoroughly assayed for sensitivity and specificity in a given application. Validation of Abs against post-translationally modified (PTM) targets is particularly challenging because it requires specifically prepared antigen. Here we describe a novel validation method using surrogate proteins in a Western blot. The surrogate protein, which we termed ‘MILKSHAKE,’ is a modified maltose binding protein enzymatically conjugated to a peptide from the chosen target that is either modified or nonmodified at the residue of interest. The certainty of the residue’s modification status can be used to confirm Ab specificity. This method also allows for Ab validation even in the absence or limited availability of treated cell lysates. We have developed a novel method for validating Abs in a Western blot using modified maltose-binding protein enzymatically conjugated to a target peptide mixed with mammalian cell lysate as the antigen. This method can be used to confirm the primary antibody’s specificity for the target sequence and as a Western blot process control specific to the primary antibody used. We will demonstrate the utility of MILKSHAKE for post-translationally modified (PTM) targets and for determining specificity at an amino acid polymorphic site, but it should be apparent that it can also be used for non-PTMs.
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影响因子：
48
作者：
Uhlen M;Bandrowski A;Carr S;Edwards A;Ellenberg J;Lundberg E;Rimm DL;Rodriguez H;Hiltke T;Snyder M;Yamamoto T
通讯作者：
Yamamoto T
影响因子：
14.8
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影响因子：
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作者：
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通讯作者：
Cole PA
影响因子：
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作者：
Bordeaux J;Welsh A;Agarwal S;Killiam E;Baquero M;Hanna J;Anagnostou V;Rimm D
通讯作者：
Rimm D
影响因子：
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作者：
Mao, HY;Hart, SA;Pollok, BA
通讯作者：
Pollok, BA