Whole hearts were homogenized in RIPA lysis buffer and the extract spun at 13200 g, 10 min, 4 °C. Total cell lysates were electrophoresed and transferred to PVDF membrane and blocked with Odyssey Blocking Buffer (Li-Cor Biosciences). The membrane was incubated with primary antibodies for 2 h at room temperature or overnight at 4˚C, washed in TBST washing solution, and incubated in Odyssey IRDye secondary antibodies (1:5000) for 1 h before visualization with the Odyssey Infrared Imaging System (Li-Cor Biosciences). The primary antibodies used: for detection of LaminA/C (Rabbit, 1:500, Cell Signaling) that is specific to an epitope in the first 50 amino acids in LMNA, mSun1 (mouse mAB, clone 12.11, neat, from B. Burke), GFP (mouse, 1:500, Roche), LaminB1 (rabbit mAB, 1:500, Abcam), anti-HA epitope (rat mAB, 3F10, 1:1000, Roche), GAPDH (rabbit, 1:500, Abcam), and control β-tubulin (mouse, Tub 2.1, 1:1000, Sigma). For detection of the AAV9-DNhSun1 transgene, a mouse mAB specific to the C-terminus of human Sun1 (hSun1, clone 9.1, neat, from B. Burke) was used in combination with protein A conjugated to HRP (1:500, Cell Signaling). GAPDH (rabbit, 1:500, Abcam) was used with anti-rabbit Immunoglobulins/HRP (DAKO, 1:2500) as a loading control. Uncropped and unprocessed scans of blots are in the Source Data file.
Gapdh
Manufactured by Abcam
Sourced in United States, United Kingdom, China, Germany, Hong Kong, Japan, Canada, Norway, Israel
GAPDH (Glyceraldehyde 3-phosphate dehydrogenase) is a key enzyme involved in the glycolytic pathway, catalyzing the reversible oxidation of glyceraldehyde-3-phosphate to 1,3-bisphosphoglycerate. It plays a crucial role in cellular energy production.
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Lab products found in correlation
Pvdf membrane, by Merck Group (347 mentions)
Bcl 2, by Abcam (177 mentions)
E cadherin, by Abcam (174 mentions)
Ripa lysis buffer, by Beyotime (166 mentions)
Vimentin, by Abcam (142 mentions)
Bcl2 associated x (bax), by Abcam (142 mentions)
N cadherin, by Abcam (140 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (134 mentions)
β actin, by Abcam (133 mentions)
Bca protein assay kit, by Beyotime (131 mentions)
Ripa buffer, by Beyotime (98 mentions)
P akt, by Abcam (78 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (76 mentions)
Caspase 3, by Abcam (75 mentions)
Ripa buffer, by Thermo Fisher Scientific (75 mentions)
Bca kit, by Thermo Fisher Scientific (74 mentions)
Cleaved caspase 3, by Abcam (72 mentions)
Ripa lysis buffer, by Thermo Fisher Scientific (71 mentions)
β catenin, by Abcam (69 mentions)
Cyclin d1, by Abcam (66 mentions)
2 872 protocols using gapdh
1
Protein Expression Analysis in Mouse Hearts
2
Immunoblotting Analysis of DNA Damage Markers
3
Quantification of Protein Expression
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Total protein was extracted using a RIPA lysis buffer (Fdbio science, Hangzhou, China). The proteins were then separated by SDS-PAGE and transferred onto polyvinylidene fluoride membrane. The membrane was blocked with 5% nonfat dried milk and incubated with MSH2 primary antibody (1:5000, Abcam, Cambridge, UK) and goat anti-rabbit secondary antibody (1:10000, Abcam, Cambridge, UK). Proteins were detected by using enhanced chemiluminescence method and imaged by GelDoc™ XR+ system (Bio-Rad, Hercules, USA). GAPDH (1:5000, Abcam, Cambridge, UK) was chosen as an internal control and the expression level of MSH2 protein was normalized by GAPDH.
4
Quantification of Protein Signaling Pathways
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Total protein was extracted from kidney and mouse NRK-52E cells using the RIPA cell lysis buffer system (Cell Signaling Technology, USA), supplemented with phosphatase inhibitors, and was quantified by BCA-protein assay kit (Beyotime Institute of Biotechnology, Shanghai, China). An aliquot (20 μg) of the proteins was separated by 10% SDS-PAGE for protein electrophoresis and transferred to a polyvinylidene fluoride (PVDF) membrane (Millipore). Following blocking in 5% nonfat dry milk for 1 h at room temperature, the membrane was incubated at 4°C overnight with primary antibodies. The following antibodies were used: mouse RIPK1, p-RIPK3/RIPK3, p-MLKL/MLKL, IL-1β, anti-active caspase-3, phosphor-inhibitor κB α (p-IκBα), phosphor-inhibitor of kappa B kinase α/β (p-IKKα/β), GAPDH (all antibodies dilution ratios are 1 : 1000; Abcam, Cambridge, UK), and rabbit p-RIPK1. After washing three times with phosphate-buffered saline with Tween-20 (PBST), the membrane was incubated with HRP-conjugated secondary antibody (1 : 2000, Abcam, Cambridge, UK) at room temperature for 1 h. Bands were quantified using ImageJ software and normalized to GAPDH.
5
Quantifying p-STAT3, STAT3, and GAPDH
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The protein levels of p-STAT3(s727) (cat: #9134, 100kDa, Cell Signaling Technology, Beverly, MA, USA), total STAT3 (cat: #9189, 100 kDa, Cell Signaling Technology) and GAPDH (#ab181602; Abcam) were measured using a Western blot system (SNAP i.d. Protein Detection System; Merck Millipore, Danvers, MD, USA). Splenocytes were harvested from BALB/c mice and then stimulated with VnP-16 (100 μg/mL) or vehicle for 2 hours, and then with IL-6 (10 ng/mL) for 1 hour. Then, whole-cell lysates were prepared. The protein concentration was measured using the BCA assay method (#23235, Thermo), and samples were separated on a 4–12% sodium dodecyl sulfate polyacrylamide gel and transferred to a nitrocellulose membrane (Amersham Pharmacia, Uppsala, Sweden). The primary antibodies p-STAT3 s727 (cat: #9134, 100kDa; Cell Signaling Technology), total STAT3 (cat: #9189, 100 kDa; Cell Signaling Technology), and GAPDH (cat: ab181602, 36kDa; Abcam) were diluted in 0.1% skim milk in Tris-buffered saline Tween-20 and incubated for 20 min at room temperature. The membrane was washed and incubated with horseradish peroxidase-conjugated secondary antibody for 20 min at room temperature. Band density was estimated by image capture densitometry.
6
Protein Expression Analysis in Cells
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Total proteins were extracted from the cells or mouse tissues with RIPA buffer (CST, Waltham, MA, USA). Protein concentrations were measured using the BCA Protein Quantification Kit (Abbkine, California, USA). Equivalent concentrations of protein were separated by SDS-PAGE (12% polyacrylamide gels) and transferred to PVDF membranes (Millipore, Waltham, MA, USA) in the subsequent step. The membranes were blocked with 5% milk and incubated overnight at 4 °C with primary antibodies for HIF-1α (Abcam, Massachusetts, USA), HSP70 (Abcam, Massachusetts, USA), IGF-2 (Abcam, Massachusetts, USA), IL-6 (Abcam, Massachusetts, USA), IL-8 (Abcam, Massachusetts, USA), VEGF (Abcam, Massachusetts, USA), and GAPDH (Abcam, Massachusetts, USA), where GAPDH served as the control. Membranes were then incubated with the corresponding secondary antibodies (Abcam, Massachusetts, USA) for 1 hour at room temperature, followed by visualization using an Odyssey CLx Infrared Imaging System.
7
DEDD and GAPDH Protein Expression
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HL cells were lysed by chilled RIPA lysis buffer (9803; Cell Signaling Technology, Inc., Danvers, MA, USA) which contained phenylmethylsulfonyl fluoride protease inhibitor. The lysates were put onto ice for half an hour, then centrifuged at 14,000 × g at 4°C for 15 min. The BCA Protein Assay kit (Thermo Fisher Scientific, Inc.) was applied to detect the protein concentration according to the manufacturer's protocol. A total of 10% sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) was used to separate the proteins and then the separated proteins were transferred onto a polyvinylidene difluoride (PVDF) membrane which was incubated at 4°C overnight with primary antibodies diluted in 5% milk. The primary antibodies were as follows: rabbit polyclonal antibodies against DEDD (dilution, 1:1,000; cat. no. ab203655; Abcam, Cambridge, MA, USA) and GAPDH (dilution, 1:1,000, cat. no. ab37168; Abcam). A secondary incubation step was performed with appropriate secondary antibody for 1 h at room temperature. The proteins were detected by a chemiluminescence method. GAPDH (Abcam) was used as an internal loading control.
8
Protein Expression Analysis in hADSCs
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Total protein separated from hADSCs using a protein extraction kit (Bio-Rad, USA), and the concentration of total protein was determined using BCA kit (Tianjin, China). Subsequently, the protein (50 μg) was separated by SDS-PAGE. Then, moved to PVDF (Millipo,USA), the membrane was incubated with blocking buffer at room temperature for 1 h. Then, incubate the first antibody GAPDH (Abcam), Aggrecan (Proteintech), SOX9 (Proteintech), COL2A1 (Proteintech), BMPR2 (Abcam) overnight, followed by secondary antibody at room temperature for 1 h. The GAPDH was used internal control. All western blot experiments were repeated at least three times.
9
Comprehensive Molecular Analysis Techniques
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RT-qPCR, Western blotting and co-immunoprecipitation (Co-IP) were performed as previously described.9 (link) The primer sequences for PCR were as follows: ROCK2 forward, 5ʹ-GGTATCTGTACATGGTAATGG-3ʹ and reverse, 5ʹ-GGAGTGTATTGCATCCAGAG-3ʹ; HKII forward, 5ʹ-GGCAATGAAACCAAAGCCAGGAG-3ʹ and reverse, 5ʹ-GGAAGGAGGAGCCAGAAGC-3ʹ; GAPDH forward, 5ʹ-CCTGCCGGTGACTAACCCTG-3ʹ and reverse, 5ʹ-AGTTAAAAGCAGCCCTGGTG-3ʹ. The antibodies of Western blot were as follow: ROCK2 (Abcam 1:1000 dilution), HKII (Abcam 1:1000 dilution), BCL-2 (Abcam 1:2000 dilution), BAX (Abcam 1:2500 dilution), AKT/P-AKT (Abcam 1:500 dilution) PI3K/P-PI3K (Abcam 1:1000 dilution), GAPDH (Abcam 1:5000 dilution).
10
Western Blot for Protein Analysis
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Western blot technique is used to transfer fragments of proteins from an electrophoresis gel to a membrane. It helps in detecting the proteins of interest expressed by the cells. Cells were lysed and the pellet centrifuged at 12,000 rpm for 15 min for western blot analysis. In the supernatant, protein was estimated, and an equal amount of protein was measured and placed onto the gel. AMPK, p-AMPK, and GAPDH antibodies (Abcam, Cambridge, UK) were employed as primary antibodies, and HRP-conjugated secondary antibodies were utilised to detect proteins of interest AMPK, p-AMPK, and GAPDH using the Western blot method.
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