Cells were lysed with ice-cold lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China), and protein was separated with 12% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (Beyotime Institute of Biotechnology), which was then transferred onto polyvinylidene difluoride membrane (Thermo Fisher Scientific). The membrane was then incubated with PBS containing 5% non-fat milk (Yili, Beijing, China) overnight at 4°C. After washed with PBST for 3 times, the membrane was incubated with rabbit polyclonal anti-SIRT1 antibody (1:200; Abcam, Cambridge, MA, USA) or rabbit polyclonal anti-GAPDH antibody (1:200; Abcam) at room temperature for 3 h. After washed with PBST for 3 times, the membrane was incubated with goat anti-rabbit secondary antibody (1:10,000; Abcam) at room temperature for 1 h. The immunoreactive band was detected using the enhanced chemiluminescence system (Thermo Fisher Scientific), according to the manufacture's instruction. The protein expression was measured using Image Pro Plus software (Media Cybernetics, Rockville, MD, USA).
Anti gapdh polyclonal antibody
Manufactured by Abcam
Sourced in United Kingdom, United States
The Anti-GAPDH polyclonal antibody is a research-use-only product. It is designed to detect and bind to the GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) protein, which is a widely expressed enzyme involved in glycolysis. The antibody can be used to study GAPDH expression and distribution in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Goat anti rabbit igg hrp conjugate, by Bio-Rad (2 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Polyvinylidene difluoride membrane, by Thermo Fisher Scientific (1 mentions)
Goat anti rabbit secondary antibody, by Abcam (1 mentions)
Enhanced chemiluminescence system, by Thermo Fisher Scientific (1 mentions)
Ice cold lysis buffer, by Beyotime (1 mentions)
Sds page, by Beyotime (1 mentions)
Image pro plus software, by Media Cybernetics (1 mentions)
Goat anti mouse igg horseradish peroxidase hrp conjugate, by Bio-Rad (1 mentions)
Bm chemiluminescence blotting substrate pod, by Roche (1 mentions)
Bca protein assay kit, by Bio-Rad (1 mentions)
Anti nrf2 polyclonal antibody, by Abcam (1 mentions)
Anti β actin polyclonal antibody, by Abcam (1 mentions)
Anti ho 1 monoclonal antibody, by Abcam (1 mentions)
Phospho y607, by Abcam (1 mentions)
Goat anti rabbit igg, by Abcam (1 mentions)
Enhanced chemiluminescence detection system, by Bio-Rad (1 mentions)
Amersham enhanced chemiluminescence ecl detection system, by GE Healthcare (1 mentions)
Pierce coomassie plus bradford assay kit, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
7 protocols using anti gapdh polyclonal antibody
1
Western Blot Analysis of SIRT1 Protein
2
Western Blot Immunodetection Protocol
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The nitrocellulose membranes were blocked with 3% BSA in Tris-buffered saline with 0.1% Tween-20 (TBS-T) for 1 h and then incubated for 1 h with primary antibody. The following primary antibodies were used: polyclonal anti-SopB antibody (1:1,000; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA), polyclonal anti-p65 antibody (1:500; Santa Cruz Biotechnology Inc.), polyclonal anti-phospho-Akt antibody (1:500; Abcam Inc., Cambridge, MA, USA), polyclonal anti-Akt antibody (1:500; Abcam Inc.), monoclonal anti-phospho-p38 MAPK (Thr180/Tyr182) (1:1000; Cell Signaling Technology, Beverly, MA, USA), polyclonal anti-p38 MAPK antibody (1:1000; Cell Signaling Technology), polyclonal anti-lamin-B antibody (1:1,000; Santa Cruz Biotechnology Inc.), polyclonal anti-GAPDH antibody (1:3,000; Abcam Inc.), and polyclonal anti-beta actin antibody (1:5000; Abcam Inc.). After washing the membranes three times with TBST, the secondary antibody of goat anti-mouse IgG horseradish peroxidase (HRP) conjugate, goat anti-rabbit IgG HRP conjugate, or rabbit anti-goat IgG HRP conjugate (1:3,000; Bio-Rad) was incubated with the membranes for 1 h. The blots were developed using a BM chemiluminescence blotting substrate (POD; Roche, Mannheim, Germany).
3
HOXA1 Protein Expression in Liver Cancer
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HOXA1 protein expression in liver cancer cell lines with the transfections was measured via western blots. The transfected liver cancer cells, HepG2 and Huh7, were lysed using lysis buffer. After centrifugation at 12,000 × g for 20 min at 4°C, a BCA protein assay kit (Bio-Rad Laboratories, Inc., Hercules, CA, USA) was used to assess the protein concentrations. Proteins were separated with 10% SDS-PAGE and then electrophoretically transferred onto PVDF membrane. After being blocked in 5% non-fat milk 1 h at room temperature, the membrane was then incubated with appropriately diluted primary antibodies [rabbit anti-HOXA1 polyclonal antibody (1:500; cat. no. ab230513; Abcam, Cambridge, UK) and anti-GAPDH polyclonal antibody (1:2,500; cat. no. ab9485; Abcam)] at 4°C overnight. Subsequently, the membranes were incubated with horse-radish peroxidase-linked secondary goat anti-rabbit polyclonal antibody (1:2,000; cat. no. ab6721; Abcam) for 1 h. The enhanced chemiluminescence (ECL-plus, Amersham Pharmacia Biotech Inc., Piscataway, NJ, USA) method was used to detect the proteins.
4
Western Blotting for Nrf2, HO-1, PI3K, and Akt
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Western blotting was performed as previously described. For the detection of Nrf2 and HO-1 protein levels, total proteins were extracted from the SKPs and 3D skin models using the lysis and protein loading buffers. The protein concentrations were determined using Pierce BCA Protein Assay. The Bis-Tris Gel system was established, and the primary antibodies against protein nuclear Nrf2 (anti-Nrf2 polyclonal antibody, 1 : 1000, Abcam, UK), HO-1 (anti-HO-1 monoclonal antibody, 1 : 2000, Abcam, UK), phosphorylated PI3K (p-PI3K, anti-PI3K polyclonal antibody, and phospho Y607, 1 : 1000, Abcam, UK), and phosphorylated Akt (p-Akt, anti-Akt polyclonal antibody, and phospho T308, 1 : 500, Abcam, UK) were introduced. After incubation with the secondary antibody (goat anti-rabbit IgG, 1 : 5000; Abcam, UK), signals were detected and analyzed using enhanced chemiluminescence detection system (Bio-Rad Laboratories Inc., CA, USA) and Image software. Anti-β-actin polyclonal antibody (1 : 5000; Abcam, UK) or anti-GAPDH polyclonal antibody (1 : 5000; Abcam, UK) was used as internal controls.
5
Quantitative Analysis of TET2 Protein
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Cells were lysed with radioimmunoprecipitation (RIPA) buffer (Sigma-Aldrich) on ice. Total protein concentration was determined using the Pierce Coomassie Plus Bradford assay kit, following the manufacturer’s instructions (Thermo Fisher Scientific). Proteins were resolved by 10% SDS-PAGE and electrotransferred onto a nitrocellulose membrane. After blocking with 5% bovine serum albumin, the membrane was incubated with rabbit anti-TET2 polyclonal antibody (1:300 dilution; Sigma-Aldrich) or anti-GAPDH polyclonal antibody (1:2,000 dilution; Abcam) overnight at 4°C. The membrane was then incubated with horseradish peroxidase (HRP)-conjugated anti-rabbit immunoglobulin G (IgG) for 1 h at room temperature. Signals were visualized with an Amersham enhanced chemiluminescence (ECL) detection system (GE Healthcare, Cambridge, MA, USA). Densitometry was performed using Quantity One software (Bio-Rad Laboratories, Hercules, CA, USA).
6
Western Blot Analysis of SMAD4
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Cells were lysed with RIPA lysis buffer supplemented with protease and phosphatase inhibitors (Roche Diagnostics, Basel, Switzerland). The concentration of protein was determined by Pierce® BCA Protein Assay kit (Thermo Fisher Scientific, Inc., Shanghai, China). The proteins were separated with 10% sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto the polyvinylidene difluoride (PVDF) membranes (EMD Millipore, Billerica, MA, USA). The membranes were blocked for 1 h with 5% bovine serum albumin in TBS at room temperature. Then we incubated the PVDF membranes with rabbit anti-SMAD4 polyclonal antibody (1:500; cat. no. ab110175; Abcam, Cambridge, UK,) and anti-GAPDH polyclonal antibody (1:1,200; cat. no. ab9485; Abcam) in TBST (0.1% Tween-20 in TBS) at 4°C overnight, followed by incubation with rabbit antibody for 2 h at room temperature. GAPDH was used as an endogenous reference for normalization. The proteins were detected using chemiluminescence method (ECL; EMD Millipore).
7
Western Blot Analysis of Cell Signaling Proteins
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Western blot analysis was performed as previously reported (Kanda et al., 2011) . Briefly, cells were lysed with Cell Lysis Buffer (Cell Signaling Technology, Danvers, MA, USA). The proteins were then separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretically transferred to Immobilon-P membrane (Millipore, Billerica, MA, USA). The membranes were probed with an anti-cdc25C monoclonal antibody (1:1,000; Cell Signaling Technology), an anti-cyclin B1 monoclonal antibody (1:1,000; Cell Signaling Technology), and an anti-GAP-DH polyclonal antibody (1:2,500; Abcam, Cambridge, UK) followed by incubation with horseradish peroxidaseconjugated secondary antibodies against rabbit or mouse IgG (Cel Signaling Technology). The bands were visualized using the ECL Western Blotting Analysis System (GE Healthcare, Buckinghamshire, UK), and images were acquired using a LAS-3000 Imager (FUJIFILM UK Ltd., Systems, Bedford, UK).
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