Whole-cell protein lysates were generated and analyzed by western blotting as described before [18 (link)]. Whole-cell protein extracts for HSP90 pull-down with geldanamycin were prepared with lysis buffer containing 50 mM HEPES, 150 mM NaCl, 1% Triton-X100, protease and phosphatase inhibitors. Lysates (250 μg) were incubated with 40 μg geldanamycin-biotin (Sigma-Aldrich) and 125 μl settled NeutrAvidin Agarose Resin (Thermo Scientific) at 4°C. Immobilized proteins were washed three times, resuspended in Laemmli buffer and subjected to SDS-PAGE and western blotting. The HSP90 pull-down with ATP beads was performed using the Pierce Kinase Enrichment Kit with ATP Probe (Thermo Scientific) according to the manufacturer's instructions. The following antibodies were used: anti-β-actin (Sigma-Aldrich), anti-RAF1 (#9422, Cell Signaling), anti-AKT1 (2H10, Cell Signaling), anti-STK33 (4F7, Abnova), anti-HSP90β (D-19, Santa Cruz), anti-HSP90α (AB3466, Millipore) and anti-p-glycoprotein (anti-ABCB1) (C219, Millipore).
Anti abcb1
Manufactured by Merck Group
Sourced in Germany
The Anti-ABCB1 is a laboratory product that functions as an inhibitor of the ABCB1 protein, also known as P-glycoprotein. The ABCB1 protein is a transmembrane efflux pump that plays a role in the transport of various substances across cellular membranes. The Anti-ABCB1 product can be used in research applications to investigate the function and regulation of the ABCB1 protein.
Automatically generated - may contain errors
Lab products found in correlation
Anti raf1, by Cell Signaling Technology (1 mentions)
Neutravidin agarose resin, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti c myc, by Cell Signaling Technology (1 mentions)
Anti β catenin, by Cell Signaling Technology (1 mentions)
Anti cyclin d1, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Bca protein assay kit, by Boster Bio (1 mentions)
Ripa lysis buffer, by Beyotime (1 mentions)
2 protocols using anti abcb1
1
Protein Interactome Profiling Using Affinity Purification
2
Protein Expression Analysis by Western Blot
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Cells were harvested and suspended in radioimmunoprecipitation assay (RIPA) lysis buffer (Beyotime, China) and quantified with a bicinchoninic acid (BCA) protein assay kit (Boster, Wuhan, China). After boiling in sample buffer, total proteins were separated by denaturing 8% or 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrotransferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). Subsequently, the membranes were then blocked and incubated with the following antibodies overnight at 4°C: anti-ABCB1 (Millipore, Merck, Germany), anti-β-catenin, anti-c-Myc, and anti-cyclin D1 (Cell Signaling Technology, USA), anti-DKK2 (Abcam, England), and anti-GAPDH (Santa Cruz Biotechnology, Santa Cruz, CA, USA) as a control. Horseradish peroxidase-conjugated anti-mouse, anti-rabbit antibodies were used as secondary antibodies correspondingly. The bands were visualized by chemiluminescence reagents (ChemiQ 4600, Bioshine).
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