Western blotting was performed as previously described23 (link). In brief, 10 mg of tissue was added to 500 µl ice cold radioimmunoprecipitation (RIPA) buffer containing a protease inhibitor (Roche) and homogenized on ice using a rotor-stator homogenizer (IKA) followed by centrifugation at 13,000 rpm at 4 °C for 30 min. Protein lysates were incubated in 4x Roti load sample buffer (Roth) for 5 min at 90 °C and resolved by SDS-PAGE (12% gels). Proteins were then transferred to PVDF membranes (Bio-Rad) using the Mini-Protean system (Bio-Rad). After blocking nonspecific binding, PVDF membranes were incubated with primary antibodies, including chicken anti-GFP (Aves Labs; 1:2000) for eGFP-L10a detection. Anti-GAPDH (Bethyl Labs, 1:5000) and anti-β-actin antibody (Santa Cruz, 1:10000) served as loading control. Membranes were subsequently probed with HRP-conjugated secondary antibody (Santa Cruz Biotechnology/ Bethyl Labs, 1:10000). Chemiluminescent detection of Proteins was performed using ClarityTM ECL substrate (Bio-Rad) on the Fusion FX (Vilber) chemiluminescent imaging platform.
Anti gapdh
Manufactured by Fortis Life Sciences
Sourced in United States
Anti-GAPDH is a laboratory reagent used for the detection and quantification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) protein. GAPDH is a commonly used internal control and housekeeping gene in various molecular biology and cell biology applications. Anti-GAPDH is a highly specific antibody that binds to GAPDH and allows for its identification and measurement in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Protease inhibitor, by Roche (2 mentions)
Anti flag, by Merck Group (2 mentions)
Anti eif3a, by Novus Biologicals (2 mentions)
Nbp1 18891, by Fortis Life Sciences (2 mentions)
Anti eif3b, by Fortis Life Sciences (2 mentions)
Anti eif3c, by Fortis Life Sciences (2 mentions)
Anti eif3e, by Fortis Life Sciences (2 mentions)
Anti eif3f, by Fortis Life Sciences (2 mentions)
Anti eif3g antibody, by Fortis Life Sciences (2 mentions)
Anti eif3h antibody, by Fortis Life Sciences (2 mentions)
Anti eif3i, by BioLegend (2 mentions)
Anti eif3k, by Novus Biologicals (2 mentions)
Anti eif3l antibody, by GeneTex (2 mentions)
Anti eif3m, by Novus Biologicals (2 mentions)
Anti eif4g1, by Fortis Life Sciences (2 mentions)
Anti eif3d, by Fortis Life Sciences (2 mentions)
Anti rps19, by Fortis Life Sciences (2 mentions)
Anti c jun, by Fortis Life Sciences (2 mentions)
Anti btg1, by Abcam (2 mentions)
Anti hsp90, by BD (2 mentions)
12 protocols using anti gapdh
1
Quantitative Western Blotting Analysis
2
Protein Extraction and Western Blot Analysis
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Cells and tissues were lysed by radioimmunoprecipitation (RIPA) buffer without sodium deoxycholate (50 mM Tris–HCl pH 7.6, 150 mM NaCl, 1% NP-40) with various inhibitors (1 mM Na3VO4, 1 mM AEBSF, 1 mM DTT, 10 μg/ml aprotinin, 1 μg/ml leupeptin, 10 mM NaF, 1 μg/ml pepstatin A) and cOmplete Protease Inhibitor Cocktail (Roche, Basel, Switzerland). Protein concentrations in the supernatants were detected by the DC™ protein assay kit (Bio-Rad Laboratories, Hercules, USA). The equal amounts of proteins were separated by SDS-PAGE. Proteins were detected by the following antibodies diluted by CanGetSignal Solution 1 (Takara Bio Inc.); anti-GAPDH (Bethyl Laboratories, Montgomery, TX, USA), anti-DYRK2 (Sigma-Aldrich), anti-phospho-Akt (Ser473) (Cell Signaling Technology, Danvers, MA, USA), anti-Myc, anti-Myc-Ser62 (Abcam), anti-FLAG (Sigma-Aldrich), anti-Hras, anti-cyclin E, anti-cyclin D1, and anti-cyclin D2 (Santa Cruz Biotechnology, Dallas, TX, USA). Membranes were developed by ImmunoStar LD (Fujifilm Wako Pure Chemical Co., Osaka, Tokyo) and imaged using FUSION SOLO 4 M, and analysed using Fusion Capt Advance software (M&S Instruments Inc., Osaka, Tokyo).
3
Comprehensive Protein Expression Analysis Using Western Blotting
4
Western Blot Analysis of Cellular Proteins
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Whole-cell lysates were prepared by incubation with whole-cell lysis buffer that included 0.5% NP40 and 1% SDS supplemented with HALT protease and phosphatase inhibitors (Sigma, St Louis, MO, USA). Lysates were cleared by centrifugation and protein concentration was tested by DC assay (Bio-Rad, Shanghai, China ). Lysates were boiled with SDS sample buffer, separated by SDS-PAGE, and transferred to polyvinylidene difluoride membrane (Millipore, Guangzhou, China). Membranes were blocked in 5% nonfat dry milk TBS-T (10 mmol/l Tris-HCl (pH 7.4), 150 mmol/l NaCl, 0.1% Tween 20) buffer and incubated in primary antibodies diluted in blocking buffer at 4 °C overnight. Blots were washed with TBS-T buffer and incubated with horseradish peroxidase-conjugated secondary antibodies (1 : 10 000; GE Healthcare, Shanghai, China) in blocking buffer at room temperature. Immune complexes were visualized with an enhanced chemiluminescence kit (GE Healthcare). Primary antibodies for immunodetection were sourced as follows: anti-tubulin (goat, Santa Cruz Biotechnology, Guangzhou, China), anti-DDX59 (Abcam, Guangzhou, China), anti-GAPDH (Bethyl, Beijing, China), anti-Lamin B1 (Santa Cruz Biotechnology), anti-SOD (Santa Cruz Biotechnology).
5
Comprehensive Protein Expression Analysis Using Western Blotting
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Western blot analysis was performed using the following antibodies: anti-eIF3a (Novus NBP1-18891); anti-eIF3b (Bethyl A301-761A); anti-eIF3c (Bethyl A300-376A); anti-eIF3d (Bethyl A301-758A); anti-eIF3e (Bethyl A302-985A); anti-eIF3f (Bethyl A303-005A); anti-eIF3g antibody (Bethyl A301-757A); anti-eIF3h antibody (Bethyl A301-754A); anti-eIF3i (Biolegend 646701); anti-eIF3k (Novus NB100-93304); anti-eIF3l antibody (Genetex GTX120119); anti-eIF3m (Novus NBP1-56654); anti-rpS19 (Bethyl A304-002A); anti-eIF4G1 (Bethyl A301-775A); anti-c-Jun (Bethyl A302-959A); anti-BTG1 (Abcam ab151740). anti-GAPDH (Bethyl A300-640A); anti-HSP90 (BD 610418).
6
Western Blot Analysis of NAMPT Protein
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Brains were dissected and homogenized in ice-cold RIPA lysis buffer (Beyotime Institute of Biotechnology). Then they were centrifuged with 8,700 x g at 4˚C for 10 min to obtain total proteins. After that each sample was quantitatively tested by Nano Drop, and loaded with the same total protein amount (50 mg). The proteins were separated by 10% SDS-PAGE, and transferred to PVDF membranes using an electrophoretic transfer system (Bio-Rad Laboratories, Inc.). Membranes were blocked against non-specific binding in 5% skimmed milk for 1 h at room temperature. The membranes were then incubated with anti-NAMPT (1:5,000; cat. no. A300-372A; Bethyl Laboratories, Inc.) or anti-GAPDH (1:1,000; cat. no. AF1186; Beyotime Institute of Biotechnology) primary antibody overnight at 4˚C. The membranes were washed by TBST (0.1% tween-20; Beyotime Institute of Biotechnology) and incubated with HRP-labeled goat anti-rabbit IgG (H+L) (1:200; cat. no. A0208; Beyotime Institute of Biotechnology for 1 h at room temperature. Then BeyoECL Moon (cat. no. P0018FS; Beyotime Institute of Biotechnology) was used for visualization. Finally, the results were analyzed using the Tanon 5200 Imaging System (Tanon Science and Technology Co., Ltd.).
7
Immunoblotting for Cellular Signaling
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Whole-cell extracts were prepared according to standard protocols and tested by western blot using anti-GAPDH (Bethyl; dilution: 1:25,000), anti-IGFBP-1 (Santa Cruz; dilution:1:1,000), anti-phospho acetyl-coA-carboxylase (Cell Signaling; dilution: 1:2,000), anti-phospho-AMPK (Cell Signaling; dilution: 1:2,000), anti-phospho-mTOR (Cell Signaling; dilution: 1:2,000), anti-phospho-p53 (Cell Signaling; dilution: 1:2,000), anti-p21 (Santa Cruz; dilution: 1:1,000), anti-gH2Ax (Millipore; dilution: 1:1,000), anti-β-actin (Sigma; dilution: 1:10,000), anti-PKM2 (Santa Cruz; dilution: 1:1,000), anti-PDHK1 (Cell Signaling; dilution: 1:2,000), OxPhos Complex Kit (Anti-Rt/ms; Invitrogen), anti-TFAM (Calbiochem; dilution: 1:1,000) and anti-PGC1α (Abcam; dilution: 1:1,000).
8
Western Blot Analysis of Prostate Cancer Markers
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Cells were lysed by lysis buffer (20 mM Tris, 150 mM NaCl, 1 mM EDTA, 0.5% Triton-X 100, protease inhibitor (Roche)) and disrupted by a sonicator. Cell lysates were centrifuged at 15,000 rpm and soluble supernatants were separated. 15–30 μg of cell lysates were mixed with sample buffer (60 mM Tris, 25% glycerol, 2% SDS, 5% β-mercaptoethanol, 0.05% bromophenol blue) and separated by 10% SDS-PAGE. The proteins were transferred to nitrocellulose membrane. Membranes of each protein size were probed with the primary antibodies and the corresponding secondary antibodies. The signals were detected using LAS-500 (GE Healthcare) according to manufacturer’s protocol. All signals were quantified using Image J software54 (link). Following antibodies were purchased: anti-AR (Cat# SC-7305, Santa Cruz Biotechnology), anti-AR-V7 specific (Cat# 68492S, Cell signaling), anti-PSA (Cat# ab53774, Abcam), anti-GAPDH (Cat# A300-641A, Bethyl), anti-Lamin A/C (Cat# 2032, Cell signaling).
9
Granulocyte Protease Inhibition for Immunoblotting
10
Western Blot Analysis of Cell Signaling
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Same numbers of cells were used to extract proteins in 1× passive lysis buffer (Promega; E194A), and protein supernatant was collected after centrifugation at 16,200 g at room temperature. Proteins were denatured in 1× sample buffer (10% glycerol, 0.002% bromophenol blue, 0.075 M Tris, 2% SDS and 5% beta-mercaptoethanol) by boiling at 99°C for 10 min and centrifuged at 16,200 g for 10 min. Western blotting was performed as described (Kumar et al, 2013 (link)). Following antibodies were used for immunoblot analyses: anti-phospho p53 (s15) (Cell Signaling; #9284), anti-phospho p38 MAPK (Thr 180/Tyr 182) (Cell Signaling; 4631), anti-phospho-YAP (Ser127) (Cell Signaling; #4911), anti-p21 (H-164) sc-756 (Santa Cruz Biotech) and anti-GAPDH (Bethyl; A300-641A).
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