Protein expression of mitochondrial fission marker, dynamin-related protein-1 (DRP-1); and fusion markers, mitofusin-1/2 (Mfn-1/2) was determined using Western blot analysis as described before (Solanki et al., 2015 (link)). Antibody dilutions: DRP-1 (ab156951, 1:1000), Mfn-1 (ab57602, 1:500) and Mfn-2 (ab56889, 1:500) were purchased from Abcam (Cambridge, MA). Anti-Secondary antibody (1:2000, Cell Signaling Technology, Danvers, MA) was used. β-actin was used as a loading control (sc-47778, 1:1000, Santa Cruz Biotechnology, Santa Cruz, CA).
Ab156951
Manufactured by Abcam
Ab156951 is a laboratory equipment product offered by Abcam. It is a device designed for use in scientific research and analysis. The core function of this product is to perform a specific task or measurement as part of experimental protocols and procedures.
Automatically generated - may contain errors
Lab products found in correlation
Ab57602, by Abcam (2 mentions)
Ab56889, by Abcam (2 mentions)
Sc 47778, by Santa Cruz Biotechnology (1 mentions)
Polyvinylidene difluoride membrane, by Merck Group (1 mentions)
Anti rabbit igg, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Ab8227, by Abcam (1 mentions)
Ecl prime, by Cytiva (1 mentions)
Imagequant las 4000, by Cytiva (1 mentions)
Ab78547, by Abcam (1 mentions)
Ab283317, by Abcam (1 mentions)
Sc 81656, by Santa Cruz Biotechnology (1 mentions)
Pierce co immunoprecipitation kit, by Thermo Fisher Scientific (1 mentions)
Ab8224, by Abcam (1 mentions)
Ab196495, by Abcam (1 mentions)
Ab170901, by Abcam (1 mentions)
Immunoblot, by Merck Group (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
4 protocols using ab156951
1
Mitochondrial Dynamics Protein Analysis
2
Mitochondrial Dynamics Protein Analysis
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H-MSCs and E-MSCs at passages 4–6 were cultured in 6-well plates for 3 days. Cells were then resuspended in lysis buffer containing 20 mm HEPES, 2 mm EGTA, 1% Triton X-100, phosphatase inhibitors (5 mM Na4P2O7, 50 mM NaF, 5 mM Na3VO4, 10 mM sodium β-glycerophosphate) and a protease inhibitor cocktail (Sigma-Aldrich). After 30 min, cells were cleared by centrifugation at 15,000 × g for 10 min at 4°C. Proteins was resolved in 9% SDS-PAGE gel and transferred to polyvinylidene difluoride membranes (Millipore). Detection of MFN1 (#ab57602, Abcam), MFN2 (#ab56889, Abcam), and DRP-1 (#ab156951, Abcam) were performed according to manufacturer instructions. Beta-actin (#ab8227, Abcam) was used as a loading control. Secondary antibody was anti-rabbit IgG (#7074, Cell Signaling Technology). Detection was performed with ECL Prime (Cytiva). Images were acquired using ImageQuant LAS 4000 (Cytiva) and analyzed in ImageJ software.
3
Coimmunoprecipitation Assay for Protein Interactions
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Coimmunoprecipitation was performed using a Thermo Scientific Pierce Coimmunoprecipitation Kit (Thermo Scientific, Rockford, AL, USA) as previously described 32 (link), 33 (link). The following antibodies were used: anti-FADD (14906-1-AP; Proteintech), anti-caspase-8 (AF6442; Affinity; sc-81656; Santa Cruz), anti-RIPK1 (A7414; ABclonal), anti-Drp1 (ab156951; Abcam; 8570S; Cell Signaling Technology), anti-Fis1 (A5821; ABclonal) and anti-TOMM20 (ab186735, ab283317, ab78547; Abcam). In addition, normal IgG was added, and the samples were incubated at 4 °C overnight on a rotator. Then, 25 µl of G/A agarose beads was added to 200 µl of lysate, and the mixture was incubated for 3 h with gentle agitation at room temperature according to the manufacturer's protocol. The beads were rinsed, boiled and then removed by centrifugation at 14,000 × g for 12 min, after which the final samples were analyzed via western blotting.
4
Quantification and Analysis of Cellular Proteins
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To obtain total cellular protein, cells and rat ovaries were treated with RIPA buffer containing protease inhibitors. The total protein was further quantified using the BCA method (Pierce, Rockford, IL, USA). A 10% SDS–polyacrylamide gel was used to separate 20 μg of total protein by electrophoresis. The proteins were transferred onto PVDF membranes (Millipore, Bedford, MA, USA). The membranes were further blocked with 5% nonfat milk and subsequently incubated with rabbit monoclonal SYVN1 antibody (Abcam, ab170901; 1 : 1,000), rabbit polyclonal cleaved-caspase-3 (Abcam, ab49822, 1 : 500), rabbit polyclonal Bax (Sigma-Aldrich, B3428, 1 : 500), rabbit polyclonal Bcl-2 (Abcam, ab196495, 1 : 500), mouse monoclonal Drp1 (Abcam, ab156951, 1 : 1,000), and rabbit polyclonal Mnf1 (Sigma-Aldrich, SAB2106161, 1 : 1,000), overnight at 4°C. Next, the membranes were incubated with respective peroxidase-conjugated secondary antibody for 1 h. Proteins bands were finally detected using an enhanced chemiluminescent detection system (Immunoblot, 23225; Millipore, Billerica, MA, USA). Band intensity of the target protein was obtained using ImageJ (NIH, USA) and normalized with that of the mouse monoclonal β-actin (Abcam, ab8224, 1 : 1,000).
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