Cells were collected and resuspended in RIPA lysis buffer for 60 min incubated on ice. After centrifuging for 20 min at 12000g and 4°C, the supernatant was collected. Protein concentrations were determined by using Bicinchoninic Acid Protein Assay kit (Thermo Fisher Scientific, Rockford, IL, USA). 60 μg of protein was separated on a 10% SDS-PAGE gel and transferred onto a polyvinylidene difluoride (PVDF) membrane (Millipore, Billerica, MA, USA). The membrane was rinsed with TBST and incubated in 5% nonfat milk diluted with TBST at 37°C for 2 h. The membrane was then incubated with mouse anti-Bak (1:1000 dilution, Cell signaling technology), mouse anti-c-PARP (1:1000 dilution, Cell signaling technology), and mouse anti-GAPDH primary antibodies (1:3000 dilution, Santa Cruz, Dallas, Texas, USA) respectively overnight at 4°C. After washing with TBST 3 times for 15 min each time, the membrane was incubated with the appropriate secondary antibodies for 1 hour at 37°C. The membranes were then washed with TBST 4 times for 10 min each time and visualized the bands by using an ECL kit (Millipore, Billerica, MA) in a Gel Doc™ XR+ and ChemiDoc™ XRS+ System (BioRad, Hercules, CA). Image-Pro plus software 6.0 (Media Cybernetics, Rockville, MD, USA) was used to analyze the relative protein expression which was normalized to GAPDH.
Mouse anti gapdh primary antibody
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Mouse anti-GAPDH primary antibodies are a laboratory reagent used to detect the presence and quantity of the GAPDH protein in biological samples. GAPDH is a commonly used housekeeping gene and is involved in glycolysis. These antibodies can be used in various immunoassay techniques, such as Western blotting, immunohistochemistry, and ELISA, to analyze GAPDH expression levels.
Automatically generated - may contain errors
Lab products found in correlation
Chloral hydrate, by Merck Group (2 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Ecl kit, by Merck Group (1 mentions)
Mouse anti bak, by Cell Signaling Technology (1 mentions)
Image pro plus software 6, by Media Cybernetics (1 mentions)
Bicinchoninic acid protein assay kit, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
Gel doc xr, by Bio-Rad (1 mentions)
Enhanced chemiluminescence kit, by Bio-Rad (1 mentions)
Secondary goat anti rabbit and goat anti mouse antibodies, by Santa Cruz Biotechnology (1 mentions)
Ecl kit, by Thermo Fisher Scientific (1 mentions)
Methyl thiazolyl tetrazolium (mtt), by Merck Group (1 mentions)
Streptomycin, by Merck Group (1 mentions)
Penicillin, by Merck Group (1 mentions)
Elastase, by Merck Group (1 mentions)
Tnf α, by Thermo Fisher Scientific (1 mentions)
Horseradish peroxidase hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti mmp 9, by Abcam (1 mentions)
Rabbit anti mmp2, by Abcam (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Beyotime (1 mentions)
6 protocols using mouse anti gapdh primary antibody
1
Western Blot Analysis of Bak and c-PARP Proteins
2
Investigating cPLA2 Expression in Brain
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To investigate the effect of B12 and ω-3 on cPLA2 expression in the brain, we performed western blotting as we recently reported. Briefly, brain samples were homogenized in RIPA buffer containing proteinase inhibitors. Total protein content was assayed using Bradford reagent, and 60 μg protein was separated on SDS-PAGE, electro-transferred onto nitrocellulose membranes, and blocked in 5% skimmed milk in TBS Tween 20. The blocked membranes were probed with rabbit anti-cPLA2 and mouse anti-GAPDH primary antibodies (Santa Cruz Biotechnology-454 and Abcam 9482, respectively). After washing, the membranes were incubated with the secondary antibodies and developed using an enhanced chemiluminescence kit (Bio-Rad, USA). The blots were scanned and intensity of the obtained bands was quantified using ImageJ (NIH, USA). The results were normalized to GAPDH and are presented as percent of control.
3
Western Blot Analysis of Protein Expressions
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Tissues or cells were solubilized in cold RIPA lysis buffer. Proteins were separated with 12% SDS-PAGE, and transferred onto a polyvinylidene difluoride (PVDF) membrane. The membrane was incubated with TBST containing 5% skimmed milk at 37°C for 2 h. The membrane was then incubated with rabbit anti-ROS, rabbit anti-FIG, and mouse anti-GAPDH primary antibodies (all from Santa Cruz Biotechnology, Inc., Santa Cruz, USA), respectively, at room temperature for 2 h. After washing with PBST 4 times for 10 min each time, the membrane was incubated with the goat anti-rabbit and goat anti-mouse secondary antibodies (Santa Cruz Biotechnology, Inc.) at 4°C overnight. After washing with PBST 4 times for 10 min each time, an ECL kit (Pierce Chemical, Rockford, IL, USA) was used to perform chemiluminent detection. Image-Pro plus software 6.0 was used to analyze the relative protein expression, represented as the density ratio versus GAPDH. GAPDH was used as an internal reference.
4
Evaluating Anti-inflammatory Agents in Cell Lines
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GSP were kindly provided by Western Animal Husbandry Co., Ltd. (Xinjiang, China). TNF-α was obtained from Peprotech (Rocky Hill, USA). Fetal Bovine Serum (FBS), Dulbecco’s Modified Eagle’s Medium (DMEM), trypsin and Ethylene Diamine Tetraacetic Acid (EDTA) were obtained from GIBCO (Grand Island, USA). Tissue-Tek O.C.T. Compound was obtained from Sakura Finetek Japan Co., Ltd. (Tokyo, Japan). Antibodies of Mac-2, MCP-1, mouse anti-GAPDH primary antibody and horseradish peroxidase (HRP)-conjugated secondary antibodies were all purchased from Santa Cruz (Dallas, USA). Rabbit anti-MMP-2, rabbit anti-MMP-9 and rabbit anti-elastin primary antibodies were purchased from Abcam (Massachusetts, USA). Elastase, chloral hydrate, penicillin, streptomycin, MTT and all other reagents were purchased from Sigma-Aldrich (Beijing, China).
5
TMEM40 Protein Expression Detection
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To detect TMEM40 protein expression, tissue samples (8 paired normal and tumor tissues) were lysed, and proteins were extracted using radioimmunoprecipitation assay buffer containing 1 mM PMSF (Beyotime Institute of Biotechnology) according to standard protocols. The protein concentrations in the supernatants were determined using a BCA Protein Assay kit (cat. no. P0006; Beyotime Institute of Biotechnology). Subsequently, ~40 µg of each total protein sample were separated by SDS-PAGE on 10% gels and transferred to nitrocellulose membranes, which were then blocked with 5% fat-free milk for 2 h at room temperature. The membranes were then washed with TBST and incubated overnight at 4°C with a mouse anti-TMEM40 primary antibody (1:500; cat. no. sc-393601; Santa Cruz Biotechnology, Inc.) and mouse anti-GAPDH primary antibody (1:1,000; cat. no. 60004-1-Ig; ProteinTech Group, Inc.). After washing, membranes were incubated with appropriate HRP-conjugated secondary antibodies (1:5,000; cat. no. sc-2357; Santa Cruz Biotechnology, Inc.) for 1 h at room temperature. The signals on the membrane were detected using an enhanced chemiluminescent reagent kit (cat. no. 36222ES60; Shanghai Yeasen Biotechnology Co., Ltd.).
6
Grape Seed Polyphenol Extraction and Characterization
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GSP were kindly provided by Western Animal Husbandry Co., Ltd. (Xinjiang, China). The GSP were extracted from grape seeds with water-ethanol double water phase system. The most abundant phenolic compounds isolated from grape seed are procyanidin, catechins, and picatechin. And content of each polyphenols is about 56.8, 5.2, and 3.6%.
Dextran sulfate sodium (DSS, molecular weight 36-50 kDa) was purchased from MP Biomedicals Co., Ltd. (Beijing, China). Antibodies of CD68 were purchased from BOSTER Biological Technology Co., Ltd. (Hubei, China). Rabbit anti-p-signal transducer and activator of transcription 3 (STAT3) and STAT3 primary antibodies were purchased from Cell Signaling Technology Co., Ltd. (MA, USA). Mouse anti-GAPDH primary antibody and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz (Dallas, TX, USA). Chloral hydrate and all other reagents were purchased from Sigma-Aldrich (Beijing, China).
Dextran sulfate sodium (DSS, molecular weight 36-50 kDa) was purchased from MP Biomedicals Co., Ltd. (Beijing, China). Antibodies of CD68 were purchased from BOSTER Biological Technology Co., Ltd. (Hubei, China). Rabbit anti-p-signal transducer and activator of transcription 3 (STAT3) and STAT3 primary antibodies were purchased from Cell Signaling Technology Co., Ltd. (MA, USA). Mouse anti-GAPDH primary antibody and horseradish peroxidase-conjugated secondary antibodies were purchased from Santa Cruz (Dallas, TX, USA). Chloral hydrate and all other reagents were purchased from Sigma-Aldrich (Beijing, China).
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