Cells were lysed and separated via the Novex NuPAGE system (Invitrogen, NP0008, NP0001, and LC3675) and the ExpressPlus PAGE Gel (GenScript). Polyvinylidene difluoride membranes (Millipore, IPVH304F0) were used for transfer and then probed with the following primary antibodies in 3% bovine serum albumin/Tris-buffered saline with 0.1% Tween 20 detergent (TBST): β-tubulin (Santa Cruz Biotechnology, sc-23949), adenosine triphosphate synthase 5 alpha (ATP5a) (Abcam, ab14748), Cyt C (Santa Cruz Biotechnology, sc-13156), MFN1 (Abcam, ab57602), MFN2 (Abcam, ab56889), and β-actin (Santa Cruz Biotechnology, sc-47778). The antibodies were detected using horseradish peroxidase–conjugated antibodies (Invitrogen, 31430), the ECL Prime Western Blotting System (GE HealthCare, RPN2232), and the Syngene Pxi Imager.
Ipvh304f0
Manufactured by Merck Group
Sourced in United States
The IPVH304F0 is a laboratory equipment product manufactured by Merck Group. It is designed to perform specific technical functions within a laboratory setting. The description of its core function is provided without any interpretation or extrapolation on its intended use.
Automatically generated - may contain errors
Lab products found in correlation
Wbkls0500, by Merck Group (3 mentions)
Ab202068, by Abcam (2 mentions)
Ab214430, by Proteintech (2 mentions)
Ecl prime western blotting system, by GE Healthcare (1 mentions)
Cyt c, by Santa Cruz Biotechnology (1 mentions)
Nupage novex system, by Thermo Fisher Scientific (1 mentions)
β tubulin, by Santa Cruz Biotechnology (1 mentions)
β actin, by Santa Cruz Biotechnology (1 mentions)
Expressplus page gel, by GenScript (1 mentions)
Iseq00010, by Merck Group (1 mentions)
Ecl reagent, by Merck Group (1 mentions)
Bca protein assay kit, by Solarbio (1 mentions)
5 protocols using ipvh304f0
1
Mitochondrial Proteome Analysis by Western Blot
2
Western Blot Analysis of Muscle Proteins
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Proteins were extracted from gastrocnemius muscles and cells, separated on 10% SDS‐polyacrylamide gels, and transferred onto PVDF membranes (Millipore IPVH304F0 and ISEQ00010, USA), which were soaked in 5% skim milk (room temperature, 1 h), washed, and then exposed overnight to primary antibodies (from Cell Signaling Technology, USA; Abcam, UK; or ProteinTech, USA): Anti‐Pim1 (D8D7Y), Anti‐GAPDH (66004‐1‐Ig), Anti‐GLB1/Beta‐galactosidase (ab128993), Anti‐p53 (ab26), Anti‐p16‐INK4A (10883‐1‐AP), Anti‐Fbx32 (ab168372), Anti‐TRIM63 (55456‐1‐AP), Anti‐PAX7 (ab199010), Anti‐Myogenin (ab1835), Anti‐MyoD1 (ab16148), Anti‐Myf5 (ab125301), Anti‐Adiponectin (ab181281), Anti‐PPAR gamma (ab45036), Anti‐CEBP Beta (ab32358), Anti‐CEBP Delta (ab65081), Anti‐FABP4 (ab92501), Anti‐SQSTM1p62 (ab109012), and Anti‐LC3B (ab192890). Subsequently, the membranes were washed and incubated with horseradish peroxidase‐labelled anti‐rabbit or anti‐mouse (ZSGB‐BIO ZB‐2305 or ZB‐2301, Beijing, China) secondary antibodies at room temperature for 90 min. After being washed, the enhanced chemiluminescence reagent (Millipore WBKLS0500, USA) was added, and the images were acquired and quantified using Image J.
3
Immunoprecipitation-based Protein Analysis
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Total proteins were extracted from gastrocnemius muscles of young and old WT mice by using an immunoprecipitation kit (Proteintech KIP2). Protein concentration was determined using a BCA Protein Assay Kit (Solarbio PC0020, China). After removing endogenous antibodies by using Protein A Sepharose beads, proteins were incubated (overnight at 4 °C) with anti‐TRB3 antibody (Proteintech 13300‐1‐AP) or IgG (Proteintech B900610), and then the antibodies and the captured proteins were precipitated using Protein A Sepharose beads and eluted using an elution buffer. The eluted proteins were separated on sodium dodecyl sulfate–polyacrylamide gels, transferred onto PVDF membranes (Millipore IPVH304F0), and probed with primary antibodies (from Abcam): Anti‐MEK1 + MEK2 (ab178876), Anti‐MEK3 + MEK6 (ab200831), Anti‐MEK4/MKK4 (ab33912), Anti‐MKK7 (ab52618), and Anti‐SQSTM1/p62 (ab109012). After incubation at 4 °C overnight, the membranes were exposed to HRP‐mouse anti‐rabbit IgG light chain‐specific secondary antibody (Proteintech SA00001‐7L), and then immunoreactive bands were visualized using the Millipore ECL reagent (WBKLS0500).
4
Immunoblotting of Muscle Apoptosis Markers
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Proteins were extracted from gastrocnemius muscles, separated on 10% sodium dodecyl sulfate–polyacrylamide gels, and transferred onto PVDF membranes (Millipore, IPVH304F0), which were soaked in Tris-buffered saline–Tween (TBST) solution containing 5% bovine serum albumin (room temperature, 1 h) to block non-specific binding; subsequently, the membranes were exposed to primary antibodies: GADPH (Proteintech, 66009-1-Ig), cleaved-caspase 9 (abcam, ab202068), cleaved-caspase3 (abcam, ab214430), Bax (Proteintech, 50599-2-Ig), Bcl2 (Proteintech 12789-1-AP). After staining overnight at 4°C, the membranes were washed thrice with TBST and incubated with HRP-labeled anti-rabbit (ZSGB-BIO, ZB-2305) or anti-mouse (ZSGB-BIO, ZB-2301) secondary antibodies at room temperature for 60 min. After washing thrice more with TBST, an enhanced chemiluminescence (ECL) reagent (Millipore, WBKLS0500) was added, and then images were acquired and quantified using ImageJ.
5
Western Blot Analysis of Apoptosis Markers
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Proteins were extracted from gastrocnemius muscles, separated on 10% sodium dodecyl sulfatepolyacrylamide gels, and transferred onto PVDF membranes (Millipore, IPVH304F0), which were soaked in Tris-buffered saline-Tween (TBST) solution containing 5% bovine serum albumin (room temperature, 1 h) to block non-speci c binding; subsequently, the membranes were exposed to primary antibodies: GADPH (Proteintech, 66009-1-Ig), cleaved-caspase 9 (abcam, ab202068), cleaved-caspase3 (abcam, ab214430), Bax (Proteintech, 50599-2-Ig), Bcl2 (Proteintech 12789-1-AP). After staining overnight at 4°C, the membranes were washed thrice with TBST and incubated with horseradish peroxidase (HRP)-labeled anti-rabbit (ZSGB-BIO ZB-2305) or anti-mouse (ZSGB-BIO ZB-2301) secondary antibodies (as appropriate) at room temperature for 60 min. After washing thrice more with TBST, an enhanced chemiluminescence (ECL) reagent (Millipore WBKLS0500) was added, and then images were acquired and quanti ed using ImageJ.
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