Protein fractions were also analyzed by SDS-PAGE denaturing gels followed by staining with SimplyBlue SafeStain solution (Invitrogen) or Western blotting as described (15 (link)). For Western blot analysis, the following primary antibodies were used: BRCA1 (C-20; Santa Cruz Biotechnology, sc-642), ubiquitin-pAb (Enzo Life Sciences, ADI-SPA-200), p53 (DO-1; Santa Cruz Biotechnology, sc-126), K63-linkage specific polyubiquitin (D7A11; Cell Signaling, #5621) and β-actin (Sigma-Aldrich, A5441). Western blot quantification and densitometry measurements were performed using Image Lab™ Software (Bio-Rad). The band intensities were selected using the volume tool. Local subtraction and linear regression methods were implemented to eliminate the local background values and to quantify the band intensities.
K63 linkage specific polyubiquitin d7a11
Manufactured by Cell Signaling Technology
K63-linkage specific polyubiquitin (D7A11) is a monoclonal antibody that recognizes polyubiquitin chains linked through Lys63 residues. It is used to detect and analyze K63-linked polyubiquitin in cellular and biochemical assays.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Simplyblue safe stain solution, by Thermo Fisher Scientific (1 mentions)
Brca1 c20, by Santa Cruz Biotechnology (1 mentions)
P53 do 1, by Santa Cruz Biotechnology (1 mentions)
β actin, by Merck Group (1 mentions)
Image lab software, by Bio-Rad (1 mentions)
Gapdh, by Aviva Systems Biology (1 mentions)
0.2 μm nitrocellulose membrane, by Bio-Rad (1 mentions)
Blotting grade blocker, by Bio-Rad (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
2 protocols using k63 linkage specific polyubiquitin d7a11
1
SDS-PAGE and Western Blot Analysis
2
Immunoblot Analysis of Protein Expression
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Immunoblot analysis was performed by standard protocol. Briefly, cells were lysed using RIPA buffer and protein concentration was determined by Pierce BCA protein assay kit (Thermo Fisher, Waltham MA). Electrophoresis and Western transfer were performed via standard protocol, and 0.2 μm nitrocellulose membrane (Bio‐Rad, Hercules, CA) was blocked using 5% Blotting Grade Blocker (Bio‐Rad). Primary antibodies for FLAG (mouse monoclonal, Sigma, St. Louis, MO) ENaCγ (rabbit polyclonal, Abcam, Cambridge, MA), K63‐linkage‐specific polyubiquitin D7A11 (rabbit monoclonal, Cell Signaling Technology, Danvers, MA),K43‐linkage polyubiquitin (rabbit polyclonal, Cell Signaling Technology) ClCN2 (mouse monoclonal, Sigma) or ClCN2 S787 (rabbit polyclonal antisera, Schiffhauer et al. 2013 (link)). Actin (rabbit polyclonal, Sigma) or GAPDH (mouse monoclonal Aviva Systems Biology, San Diego, CA) were added. Secondary antibodies included ECL™ Anti‐Rabbit or Anti‐Mouse IgG Horseradish Peroxidase linked antibody from donkey (GE Healthcare Life Sciences, Pittsburgh, PA).
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