β-Actin as a loading control: Less than 2 μg of total protein should be loaded
For the purpose of data normalization in Western blot analysis, journal editors and reviewers usually require authors to reprobe the Western blot membrane with a β‐actin‐specific antibody after detecting the target protein. In most cases, however, β‐actin is overloaded, which results in a failure to...
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| Veröffentlicht in: | Electrophoresis 2015-09, Vol.36 (17), p.2046-2049 |
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| container_title | Electrophoresis |
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| creator | Chen, Wei Xu, Wei-Hua |
| description | For the purpose of data normalization in Western blot analysis, journal editors and reviewers usually require authors to reprobe the Western blot membrane with a β‐actin‐specific antibody after detecting the target protein. In most cases, however, β‐actin is overloaded, which results in a failure to detect differences in protein loading. In this study, we attempted to optimize the amount of protein loaded for β‐actin detection to permit suitable Western blot analysis data normalization. Our data suggest that less than 2 μg of total protein should be loaded when β‐actin is used as a loading control. We also suggest avoiding reprobing the membrane with a β‐actin‐specific antibody. |
| doi_str_mv | 10.1002/elps.201500138 |
| format | Article |
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In most cases, however, β‐actin is overloaded, which results in a failure to detect differences in protein loading. In this study, we attempted to optimize the amount of protein loaded for β‐actin detection to permit suitable Western blot analysis data normalization. Our data suggest that less than 2 μg of total protein should be loaded when β‐actin is used as a loading control. We also suggest avoiding reprobing the membrane with a β‐actin‐specific antibody.</description><identifier>ISSN: 0173-0835</identifier><identifier>EISSN: 1522-2683</identifier><identifier>DOI: 10.1002/elps.201500138</identifier><identifier>PMID: 26033488</identifier><language>eng</language><publisher>Germany: Blackwell Publishing Ltd</publisher><subject>Actins - analysis ; Antibodies ; Blotting, Western - methods ; Blotting, Western - standards ; Editors ; Electrophoresis ; Failure ; Linear Models ; Linearity range ; Loading control ; Membranes ; Proteins ; Reference Standards ; Sensitivity and Specificity ; Western blot analysis ; β-Actin</subject><ispartof>Electrophoresis, 2015-09, Vol.36 (17), p.2046-2049</ispartof><rights>2015 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim</rights><rights>2015 WILEY-VCH Verlag GmbH & Co. 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| ispartof | Electrophoresis, 2015-09, Vol.36 (17), p.2046-2049 |
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| language | eng |
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| source | MEDLINE; Wiley Online Library Journals Frontfile Complete |
| subjects | Actins - analysis Antibodies Blotting, Western - methods Blotting, Western - standards Editors Electrophoresis Failure Linear Models Linearity range Loading control Membranes Proteins Reference Standards Sensitivity and Specificity Western blot analysis β-Actin |
| title | β-Actin as a loading control: Less than 2 μg of total protein should be loaded |
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