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 raf1
Manufactured by Cell Signaling Technology
Sourced in China
Anti-RAF1 is a primary antibody that recognizes the RAF1 protein, also known as c-Raf or Raf-1. RAF1 is a serine/threonine-protein kinase that plays a crucial role in the MAPK/ERK signaling pathway. This antibody can be used to detect and analyze the expression and localization of RAF1 in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti hdlbp, by Proteintech (2 mentions)
Anti abcb1, by Merck Group (1 mentions)
Neutravidin agarose resin, by Thermo Fisher Scientific (1 mentions)
Anti β actin, by Merck Group (1 mentions)
Anti p70s6k, by Cell Signaling Technology (1 mentions)
Horseradish peroxidase conjugated goat anti rabbit igg, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Cell Signaling Technology (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Protein extraction kit, by Keygen Biotech (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Anti flag, by Proteintech (1 mentions)
Anti erk, by Cell Signaling Technology (1 mentions)
Anti lamin b1, by Proteintech (1 mentions)
Anti myc, by Proteintech (1 mentions)
Anti nedd4l, by Proteintech (1 mentions)
Anti p erk, by Cell Signaling Technology (1 mentions)
Anti mek, by Cell Signaling Technology (1 mentions)
Anti p mek, by Cell Signaling Technology (1 mentions)
Anti ha, by Proteintech (1 mentions)
6 protocols using anti raf1
1
Protein Interactome Profiling Using Affinity Purification
2
Quantitative Western Blot Analysis
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Proteins were extracted from cells using a protein extraction kit (KeyGEN), and protein concentrations were measured using a BCA protein assay kit (Pierce). The proteins were then separated on sodium dodecyl sulphate‐polyacrylamide (SDS‐PAGE) gels, transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore) and probed with specific primary antibodies overnight, including anti–Raf‐1 (Cell Signalling Technology), anti‐p70S6K (Cell Signalling Technology) and the internal control anti–β‐actin (Cell Signalling Technology) or anti‐GAPDH (Santa Cruz Biotechnology). Horseradish peroxidase‐conjugated goat anti‐rabbit IgG (Cell Signalling Technology) was used as the secondary antibody. The target proteins were visualized using the Luminata Crescendo Western HRP substrate Kit (Millipore), and the integrated optical density (IOD) of target bands was quantified using Image‐Pro Plus software (Media Cybernetics).
3
Immunoblotting and CHX Chase Assays
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Immunoblotting was performed as previously described.19 (link),34 (link) The β-actin antibody was used to normalize protein expression. For CHX chase assays, cells were treated with 10 μmol CHX for 24 hours after transfection and collected at the indicated time points, and cell lysates were subjected to immunoblotting. Anti-HDLBP (Proteintech, 15406-1-AP, Wuhan, China, 1:800), Anti-RAF1 (Cell Signaling Technology, #53745, Danvers, MA, 1:800), Anti-β-actin (Proteintech, 66009-1-Ig, 1:2500), Anti-Lamin B1 (Proteintech, 12987-1-AP, 1:2500), Anti-Flag (Proteintech, 20543-1-AP, 1:2000), Anti-HA (Proteintech, 51064-2-AP, 1:3000), Anti-His (Proteintech, 66005-1-Ig, 1:2500), Anti-Myc (Proteintech, 16286-1-AP, 1:800), Anti-TRIM71 (Proteintech, 55003-1-AP, 1:800), Anti-NEDD4L (Proteintech, 13690-1-AP, 1:800), Anti-p-MEK (Cell Signaling Technology, #8727, 1:500), Anti-MEK (Cell Signaling Technology, #9154, 1:500), Anti-p-ERK (Cell Signaling Technology, #4695, 1:500), Anti-ERK (Cell Signaling Technology, #4377, 1:500), Anti-MEKK1 (Proteintech, 19970-1-AP, 1:500), Anti-RAF1S338 (Cell Signaling Technology, #9427, 1:500), Anti-RAF1S259 (Cell Signaling Technology, #9421, 1:500), Anti-RAF1S621 (Abcam, ab157201, Cambridge, UK, 1:500), Anti-RAF1Y341 (Abcam, ab59223, 1:500), and Anti-RAF1S289/296 (Cell Signaling Technology, #9431, 1:500) were used.
4
Coimmunoprecipitation Assay Protocol
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The CoIP assay was performed using CoIP kits (Abs955, Absin, Shanghai, China) according to the manufacturer’s recommended protocol. Briefly, the indicated cells were homogenized in immunoprecipitation lysis buffer (20 mM Tris-HCl pH 7.5, 0.5% NP-40, 250 mM NaCl, 3 mM EDTA, 3 mM EGTA, 1 mM DTT, 1 mM cocktail, 1 mM phosphoSTOP, 1 mM NEM, and 1 mM NAM). Five hundred micrograms of extracts were incubated with the indicated primary antibody or IgG as a negative control for 4 hours and Protein A/G-Sepharose beads for 2 hours at 4 °C. After extensive washes with phosphate buffered saline, the immunoprecipitates were used in the subsequent assays. Anti-HDLBP (Proteintech, 15406-1-AP, Wuhan, China, 1:100), Anti-RAF1 (Cell Signaling Technology, #53745, MA, USA, 1:50) and Anti-IgG (Cell Signaling Technology, #3900, MA, USA, 1:500) were used.
5
Ovarian Cancer Cell Line Characterization
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Ovarian cancer cell lines (SK-OV-3, OVCAR3 and A2780) were purchased from Type Culture Collection of Chinese Academy of Sciences and maintained according to their recommendations. The ERK inhibitor PD98059 was obtained from Sigma. Antibodies were obtained from the following companies: anti-RASAL2 antibody from Abcam; anti-β-actin antibody from Sigma; anti-E-cadherin, anti-vimentin, anti-claudin-1, anti-N-cadherin, anti-ZO-1, anti-Snail, anti-Slug, anti-Raf-1, anti-p-Raf-1, anti-MEK1/2, anti-p-MEK1/2, anti-ERK1/2 and anti-p-ERK1/2 antibodies from Cell Signaling.
6
Protein Identification via PAGE-Western Blot
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