Proteins were isolated using lysis buffer (10 mM Tris-HCl pH 7.5, 150 mM NaCl, 2 mM EDTA, 1% Triton X-100, 10% glycerol) supplemented with 1X cOmplete Proteinase Inhibitor Cocktail and 1X PhosSTOP Phosphatase Inhibitor Cocktail (both from Roche Diagnostics, Mannheim, Germany). Lysates were incubated on ice for 30 min and afterwards centrifuged at 21,250× g for 30 min at 4 °C. Western blot was performed as described elsewhere [47 (link)]. Antibodies used were anti-CD90/THY1 antibody (ab92574, Abcam, Cambridge, UK); anti-UCP1 (MAB6158, R&D, Minneapolis, MN, USA); hFAB rhodamine anti-actin and anti-tubulin (#12004164 and #12004166, BioRad).
Hfab rhodamine anti actin
Manufactured by Bio-Rad
Sourced in United States
HFAB rhodamine anti-actin is a fluorescently labeled antibody that binds to actin, a structural protein found in the cytoskeleton of cells. This product can be used to detect and visualize actin in various cell-based applications.
Automatically generated - may contain errors
Lab products found in correlation
Complete proteinase inhibitor cocktail, by Roche (1 mentions)
Anti tubulin, by Bio-Rad (1 mentions)
Ab92574, by Abcam (1 mentions)
Anti nf κb p65, by Santa Cruz Biotechnology (1 mentions)
Anti rabbit irdye 800cw, by LI COR (1 mentions)
Anti mouse irdye 680cw, by LI COR (1 mentions)
Anti pkcθ, by Cell Signaling Technology (1 mentions)
Anti erk1 2, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Cell Signaling Technology (1 mentions)
Anti flag m2, by Merck Group (1 mentions)
Anti cd3ε, by Cell Signaling Technology (1 mentions)
Anti fos, by Cell Signaling Technology (1 mentions)
Anti rat hrp, by Abcam (1 mentions)
Ne per nuclear and cytoplasmic extraction reagent, by Thermo Fisher Scientific (1 mentions)
Criterion tgx stain free protein gel, by Bio-Rad (1 mentions)
Irdye 800cw secondary antibody, by LI COR (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
Mouse anti flag m2, by Merck Group (1 mentions)
Anti rabbit igg starbright blue 700, by Bio-Rad (1 mentions)
4 protocols using hfab rhodamine anti actin
1
Protein Isolation and Western Blot Analysis
2
Immunoblotting Antibody Panel
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Anti-Centaurin alpha1 G-4 (ADAP1) (Santa Cruz, sc-390498) (1:1000); Anti-p65/NF-κB (Santa Cruz, sc-372) (1:4000); Anti-phos-ERK1/2 Thr202/Tyr204 (Cell Signaling Technology, 4370) (1:1000); Anti-ERK1/2 (Cell Signaling Technology, 4696 or 4695) (1:2000); Anti-Fos (Cell Signaling Technology, 2250) (1:1000); Anti-Jun (Cell Signaling Technology, 9165) (1:1000); Anti-Flag M2 (Sigma, F31165) (1:10,000); Anti-PKCθ (Cell Signaling Technology, 13643) (1:5000); Anti-CD3ε (Cell Signaling Technology, 4443) (1:5000); Anti-GAPDH (Cell Signaling Technology, 2118) (1:5000); hFAB Rhodamine anti-Actin (Bio-Rad, 12004166); (1:10,000); Anti-StrepTactin-HRP (Bio-Rad, 161-0381) (1:10,000); Anti-rabbit IRDye 800CW (Licor, 926-32211) (1:10,000); Anti-mouse IRDye 680CW (Licor, 925-68072) (1:10,000); Anti-mouse HRP (Cell Signaling Technology, 7076) (1:10,000); Anti-rabbit HRP (Cell Signaling Technology, 7074), (1:10,000); Anti-rat HRP (Abcam, ab97057) (1:10,000).
3
Nuclear and Cytoplasmic Protein Extraction
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The cells were harvested using trypsin-EDTA and centrifuged at 500 X g for 5 minutes, and the cell pellet was washed with PBS. The dry cell pellet was then processed for nuclear and cytoplasmic protein extraction as per manufacturer’s protocol (Thermo Scientific™NE-PER™ Nuclear and Cytoplasmic Extraction Reagents, #78833). For western blotting assay, 10 ug of the nuclear extract protein were loaded on the 4–15% Criterion™ TGX Stain-Free™ Protein Gel (Bio Rad #5678083). The protein was transferred to the nitrocellulose (Bio Rad # 1704271) using a turbo-transfer system, blocked with 1X TBST with 5% non-fat milk for an hour at 4°C, and then incubated with Anti-KAT6A / MOZ antibody (Active Motif, at 1:500) overnight. The blots were then washed and detected with IRDye® 800CW secondary antibodies (LI-COR #926-32210 and # 926-32213). For control antibody, the blots were incubated in and hFAB™ Rhodamine Anti-Actin (BioRad #12004163 at 1:2000 dilution). All experiments were performed in triplicate.
4
Immunostaining with Antibodies and Dyes
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