Total proteins of cells and exosomes were extracted, and protein concentration was determined using a BCA kit (AmyJet Scientific, Wuhan, Hubei, China). The extracted proteins were mixed with the loading buffer, boiled at 95°C for 10 min, centrifuged, electrophoresed with 10% polyacrylamide gel, and transferred to the membrane, followed by 1-h blocking with 5% skim milk in Tris-buffered saline with Tween 20 (TBST). Then, ATF2 primary antibody (1:1,000, Cell Signaling Technology, Beverly, MA, USA) was added, along with CD68 primary antibody (1:200, Abcam, Cambridge, MA, USA), CD206 primary antibody (1:200, Santa Cruz Biotechnology, Santa Cruz, CA, USA), CD63 primary antibody (1:200, Santa Cruz Biotechnology), CD81 primary antibody (1:200, Santa Cruz Biotechnology), TSG101 primary antibody (1:1,000, Abcam), GRP49 primary antibody (1:1,000, Abcam), and GAPDH primary antibody (1:1,000, Cell Signaling Technology) for overnight incubation at 4°C. Then, the membranes were developed using enhanced chemiluminescent reagent (Thermo Fisher Scientific). Quantification of signals on western blots was conducted using National Institutes of Health ImageJ imaging and processing analysis software with signaling intensity normalized to GAPDH. The experiment was repeated three times with the data averaged.
Gapdh primary antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The GAPDH primary antibody is a tool used for the detection and quantification of the GAPDH protein, which is a key enzyme involved in the glycolytic pathway. The antibody specifically binds to the GAPDH protein, allowing researchers to identify its presence and measure its levels in biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Clarity western ecl substrate, by Bio-Rad (2 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (2 mentions)
Enhanced chemiluminescent reagent, by Thermo Fisher Scientific (1 mentions)
Cd68 primary antibody, by Abcam (1 mentions)
Cd81 primary antibody, by Santa Cruz Biotechnology (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Ripa lysis buffer, by Merck Group (1 mentions)
Bicinchoninic acid kit for protein determination, by Merck Group (1 mentions)
Horseradish peroxidase conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Np 40 buffer, by Beyotime (1 mentions)
Ecl detection kit, by Beyotime (1 mentions)
Fetal bovine serum (fbs), by Santa Cruz Biotechnology (1 mentions)
Horseradish peroxidase labeled secondary antibody, by Merck Group (1 mentions)
Annexin 5 fitc, by Beyotime (1 mentions)
Caspase 3 activity detection kit, by Beyotime (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
C digit blot scanner, by LI COR (1 mentions)
Westernsure premium chemiluminescent substrate, by LI COR (1 mentions)
12 protocols using gapdh primary antibody
1
Quantitative Protein Analysis in Cells and Exosomes
2
Western Blot Analysis of FOXO3A Protein
3
Western Blot Analysis of RFC3 Protein
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The protein was extracted using NP-40 buffer (Beyotime Institute of Biotechnology, Haimen, China), and the concentration was determined using the Bicinchoninic Acid Kit for Protein Determination (Sigma-Aldrich, St. Louis, MO, USA). Samples containing 50 µg of protein were separated on a 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose membranes (Bio-Rad Laboratories, Inc., Hercules, CA, USA). The primary antibody anti-RFC3 was purchased from Santa Cruz Biotechnology, Dallas, TX, USA (Cat. no: sc-390293; dilution: 1:1,000). The membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibody (Cat. no: sc-516102; dilution: 1:10,000; Santa Cruz Biotechnology), following visualization with chemiluminescence (Thermo Fisher Scientific, Inc.). GAPDH primary antibody (Cat. no: 2118; dilution: 1:2,000; Cell Signaling Technology, Inc., Danvers, MA, USA) was used as the control antibody.
4
Optimized Endothelial Cell Culture and Analysis
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SAL was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd. (purity ≥98%; cat. no. B19208). Endothelial cell culture medium (cat. no. 26219) and cell cryopreservation solution (cat. no. 19671) were purchased from ScienCell Research Laboratories, Inc. VE-cadherin (1:1,000; cat. no. 2500), α-smooth muscle actin (1:1,000; α-SMA; cat. no. 19245), KLF4 (1:1,000; cat. no. 4038) and eNOS (1:1,000; cat. no. 9572) primary antibodies were purchased from Cell Signaling Technology, Inc. The GAPDH primary antibody (cat. no. MB001) was purchased from Bioworld Technology, Inc. The BCA protein quantitative kit (cat. no. P0010) was purchased from Beyotime Institute of Biotechnology, while the ECL detection kit (cat. no. 1829501) was purchased from MilliporeSigma and small interfering RNA (siRNA/si) was purchased from Shanghai Gema Gene Technology Co., Ltd.
5
MTT Assay for Cell Viability
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3-(4, 5-dimethyl-2-thiazolyl)-2, 5-diphenyl-2-H-tetrazolium bromide (MTT) was purchased from Beyotime Biotechnology Co. FoxM1 plasmid was from the plasmid library in our laboratory. FoxM1 primary antibody and horseradish peroxidase-labeled secondary antibody were from Sigma. GAPDH primary antibody was bought from Cell Signaling. Cell culture medium RPMI-1640 and the fetal bovine serum were purchased from Santa Cruz. K562 cells were provided by the American type culture collection (ATCC). RNA extraction and reverse transcription RT-PCR kit was purchased from Beijing Dingguo Changsheng Biotechnology Co., LTD. FITC-Annexin V and caspase 3 activity detection kits were from Beyotime Biotechnology Research Institute. MiR-29 and scramble miRNA were designed and produced by Sangon.
6
Quantifying Phosphorylated Smad Proteins
7
Molecular Mechanisms of Cardiac Fibrosis
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Deoxycortisol and MTT powder were purchased from Sigma-Aldrich Corporation. Spironolactone tablets were purchased from Hangzhou Minsheng Pharmaceutical Group Co., Ltd. GAPDH primary antibody was purchased from CST (Cell Signaling Technology. Lnc). CTGF and 11β-HSD primary antibodies were purchased from Abcam. All secondary antibodies were purchased from Shanghai Yeasen Biotechnologies Co., Ltd. Creatine kinase MB isoenzyme Assay Kit and Lactate dehydrogenase assay kit were purchased from Nanjing Jiancheng Bioengineering Institute. Masson’s Trichrome Stain Kit was purchased from Beijing Solarbio Science and Technology Co., Ltd.
8
Western Blot Analysis of Synaptotagmin-1
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Cortical neurons (14-19 DIV) expressing WT or mutant forms of syt1 were treated with lysis buffer (50 mM Tris pH 8.0, 150 mM NaCl, 2% SDS, 0.1% Triton X-100, 10 mM EDTA) supplemented with protease inhibitors (complete mini EDTA-free, Roche, 1 tablet / 50 mL lysis buffer). The lysed neurons were mixed with 4X Laemmli sample buffer and heated at 70°C for 10 minutes (stored at -20°C until use). The total proteins were separated by SDS-PAGE gel, and then transferred to PVDF membrane. The membranes were blocked with 5% milk and incubated with an anti-syt1 primary antibody (3 µg / ml; Developmental Studies Hybridoma Bank; Cat#: mAb 48) and a GAPDH primary antibody (1:1000; Cell signal; Catalog # 2118) in TBS-T with 1% milk at 4°C overnight. After washing in TBS-T, blots were incubated with Goat anti-Mouse IgG2b Cross-Adsorbed Secondary Antibody, HRP (1:2000; Invitrogen; Catalog # M32407) or Goat Anti-Rabbit IgG (H L)-HRP Conjugate (1:1000; Bio-Rad; Catalog # 172-1019) in TBS-T for 1 hr at room temperature, washed in TBS-T, and then imaged with a CCD gel imaging device (GE).
9
Western Blot Analysis of PARP and GAPDH
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For each time point, approximately 1 × 106 cells were lysed in RIPA buffer. The total protein concentration was assessed using a Pierce™ BCA Protein Assay Kit (Thermo Fisher Scientific). Laemmli buffer was added, and the samples were separated by SDS-PAGE. The proteins were transferred onto polyvinylidene fluoride membranes, and the membranes were blocked with 5% milk in TBS-Tween for 1 h. The membranes were then incubated with a 1:1000 dilution of PARP and GAPDH primary antibodies (both from Cell Signaling Technology) in TBS-Tween with 5% milk at 4 °C overnight. Subsequently, the membranes were incubated with the secondary antibody (1:5000 anti-rabbit HRP; Cell Signaling Technology) for 1 h at room temperature, and the blots were developed using the Clarity™ Western ECL Substrate (Bio-Rad Laboratories, Hercules, CA, USA) according to the manufacturer’s instructions.
10
Dissolution and Antibody Preparation Protocol
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