Cells were lysed in RIPA buffer (Sigma-Aldrich) supplemented with Complete Protease Inhibitor Cocktail (Roche). Equal amounts of proteins were prepared with NuPAGE LDS Sample Buffer and Reducing Agent (Life Technologies), separated on NuPAGE Novex 3–8% Tris-Acetate Gels (Life Technologies), and transferred on nitrocellulose membranes (Bio-Rad Laboratories, Inc.). After blocking in 5% milk and 0.1% Tween-20/TBS, membranes were incubated with primary antibodies overnight and then with HRP-conjugated secondary antibodies for 1 h. Specific signals were detected with a chemiluminescence system (GE Healthcare). The following primary antibodies were used: rabbit APC (provided by K. Neufeld, University of Kansas, Lawrence, KS) and mouse HSC70 (Santa Cruz Biotechnology, Inc.).
Mouse hsc70
Manufactured by Santa Cruz Biotechnology
Sourced in United Kingdom
Mouse HSC70 is a laboratory reagent used in research applications. It functions as a heat shock protein, which is involved in the folding and unfolding of proteins within cells. This product is intended for research use only and its specific applications should be determined by the end user.
Automatically generated - may contain errors
Lab products found in correlation
Nupage novex 3 8 tris acetate gel, by Thermo Fisher Scientific (1 mentions)
Nupage lds sample buffer and reducing agent, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Complete protease inhibitor cocktail, by Roche (1 mentions)
Mouse α tubulin, by Merck Group (1 mentions)
Enhanced chemiluminescence, by Thermo Fisher Scientific (1 mentions)
Mouse hsp70, by Santa Cruz Biotechnology (1 mentions)
Mouse hsp72, by Enzo Life Sciences (1 mentions)
Rabbit γ tubulin, by Merck Group (1 mentions)
Anti cyp4a, by Abcam (1 mentions)
3 protocols using mouse hsc70
1
Western Blot Analysis of APC and HSC70
2
Immunoblotting analysis of mitotic regulators
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Cell lysis, SDS-PAGE, and Western blotting were as previously described (Hames et al., 2005 (link)). For Western blotting, primary antibodies used were 1 µg/ml rabbit Nek6 (O’Regan and Fry, 2009 (link)), 1 µg/ml rabbit Nek7 (O’Regan and Fry, 2009 (link)), 0.3 µg/ml mouse α-tubulin (Sigma-Aldrich), 0.5 µg/ml mouse Flag (Sigma-Aldrich), 0.5 µg/ml mouse Hsp70 (Santa Cruz Biotechnology, Inc.), 0.5 µg/ml mouse Hsp72 (Enzo Life Sciences), 0.5 µg/ml mouse Hsc70 (Santa Cruz Biotechnology, Inc.), 1 µg/ml rabbit pHsp70 (this study), 0.8 µg/ml goat Nek9 (Santa Cruz Biotechnology, Inc.), 1 µg/ml rabbit GAPDH (Cell Signaling Technology), 1 µg/ml mouse phospho–histone H3 (EMD Millipore), 0.2 µg/ml rabbit γ-tubulin (Sigma-Aldrich), 0.4 µg/ml mouse TACC3 (Abcam), 0.5 µg/ml rabbit ch-TOG (Bethyl Laboratories, Inc.), and 1 µg/ml mouse Cep55 (Santa Cruz Biotechnology, Inc.). Nek7 antibodies were raised against a peptide corresponding to amino acid residues 12–28 conjugated to keyhole limpet hemocyanin via an N-terminal cysteine (C-OVPQFQPQKALRPDM). Secondary antibodies were HRP-labeled anti–mouse, anti–rabbit, or anti–goat IgGs (1:1,000; Sigma-Aldrich) or alkaline phosphatase–conjugated IgGs (1:7,500; Promega). HRP-labeled blots were detected by enhanced chemiluminescence (Thermo Fisher Scientific).
3
Renal Cortex Protein Extraction and Analysis
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Homogenates from the frozen renal cortex were prepared in 500 µL lysis buffer containing 0.1% sodium dodecyl sulfate (SDS), 0.5% sodium deoxycholate, 150 mM sodium chloride, 50 mM Tris-hydrochloride, 100 mM EDTA, 1% Tergitol (NP40), 100 mM PMSF, 1× of protease inhibitor cocktail containing aprotinin and leupeptin and phosphatase inhibitors cocktail (Bio-world). Homogenates were incubated for 1 h at 4 °C and centrifuged at 10,000× g for 30 min at 4 °C. Proteins concentrations in the supernatants were measured using the Lowry quantification method (Bio-rad Laboratory, Hercules, CA, USA). For immunoblotting, proteins (40 μg) were separated on 12.5% polyacrylamide SDS-gel electrophoresis and transferred to nitrocellulose membranes. Blots were incubated with rabbit anti-CYP4A (1:500; Abcam, Cambridge, UK), and Mouse HSC-70 (1:1000; Santa Cruz Biotechnology, Dallas, TX, USA) was used as a loading control. The primary antibodies were detected using horseradish peroxidase-conjugated IgG. Enhanced chemiluminescence helped in visualizing the bands. Densitometric analysis was performed using Image J software (1.53 e, U.S. National Institutes of Health, Bethesda, MD, USA).
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