Protein Extraction and Western Blotting

Proteins were extracted using urea buffer and analyzed by Western blotting as previously described in Pickard et al. (19 (link)). Primary antibodies used were mouse mAb to BiP (1:1000; sc-376768; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit pAb to collagen-I (1:500; OARA02579; Gentaur, Kampenhout, Belgium), mouse mAb to glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 1:10,000; clone GAPDH-71.1; MilliporeSigma), mouse mAb to vinculin (1:800; V9131; MilliporeSigma), and mouse mAb to β-actin (1:2000; sc-8432; Santa Cruz Biotechnology).

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Pickard A., Chang J., Alachkar N., Calverley B., Garva R., Arvan P., Meng Q.J, & Kadler K.E. (2019). Preservation of circadian rhythms by the protein folding chaperone, BiP. The FASEB Journal, 33(6), 7479-7489.

Publication 2019

sc-376768 V9131 sc-8432

Actin Antibodies Buffer Clone Collagen i Gapdh Glyceraldehyde 3 phosphate dehydrogenase Mouse Proteins Rabbit Urea Vinculin

Corresponding Organization : Manchester Academic Health Science Centre

Other organizations : University of Michigan–Ann Arbor

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Variable analysis

independent variables

Protein extraction using urea buffer

dependent variables

Protein expression levels measured by Western blotting

control variables

Primary antibodies used for Western blotting: mouse mAb to BiP, rabbit pAb to collagen-I, mouse mAb to GAPDH, mouse mAb to vinculin, and mouse mAb to β-actin

controls

Positive control: Not explicitly mentioned
Negative control: Not explicitly mentioned

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