Protein was extracted in RIPA buffer with proteinase and phosphatase inhibitor cocktail (ChemCruz). 20 μg of denatured total protein for each sample was loaded into an SDS-PAGE gel and separated by electrophoresis. The gel was transferred onto PVDF membranes using a wet transfer method. After transfer, the membranes were probed with the following primary antibodies against NDUFB6, 1:10,000 (ab110244, Abcam); p-PDH (Ser293), 1:1000 (AP1062, Millipore); IP3R 1:500 (sc-271197, Santa Cruz), GRP75 1:500 (sc-133137, Santa Cruz), and VDAC1 1:500 (sc-390996, Santa Cruz). Total protein was quantified from each sample using a No-Stain labeling reagent (Invitrogen) and subsequently used for total protein normalization (TPN). Anti-mouse or anti-rabbit secondary antibodies (1:10,000, Invitrogen) were incubated for 1 h at room temperature, and membranes were developed using SuperSignal West Pico PLUS or West Atto ECL (Thermo Scientific).
Sc 390996
Manufactured by Santa Cruz Biotechnology
Sourced in United States
Sc-390996 is a laboratory instrument designed for scientific research purposes. It functions as a centrifuge, which is a device used to separate different components of a liquid mixture based on their relative densities. The core function of this product is to facilitate the separation and analysis of various biological samples, such as cells, proteins, or other biomolecules.
Automatically generated - may contain errors
Lab products found in correlation
Ab110244, by Abcam (1 mentions)
Proteinase and phosphatase inhibitor cocktail, by Santa Cruz Biotechnology (1 mentions)
Ap1062, by Merck Group (1 mentions)
Sc 133137, by Santa Cruz Biotechnology (1 mentions)
West atto ecl, by Thermo Fisher Scientific (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
Prolong diamond antifade mountant, by Thermo Fisher Scientific (1 mentions)
Lsm 510 confocal microscopy, by Zeiss (1 mentions)
Alexa fluor 555 goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Immunofix, by Bio-Optica (1 mentions)
Ab110411, by Abcam (1 mentions)
Novex, by Thermo Fisher Scientific (1 mentions)
Odyssey clx near infrared imager, by LI COR (1 mentions)
Trans blot turbo transfer system, by Bio-Rad (1 mentions)
Clarity max western ecl substrate luminol solution, by Bio-Rad (1 mentions)
Sc 30141, by Santa Cruz Biotechnology (1 mentions)
Ab191838, by Abcam (1 mentions)
Tbs tween 20, by Merck Group (1 mentions)
Eclipse ci l, by Nikon (1 mentions)
5 protocols using sc 390996
1
Western Blot Analysis of Mitochondrial Proteins
2
Immunofluorescence Assay for GAC and VDAC1
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Cells (2 × 105) were plated on a coverslip and after 24 h fixed for 15 min with 4% paraformaldehyde (Immunofix, Bio-Optica, Milan, Italy; 05-K01015) in PBS, washed twice in PBS and permeabilized for 10 min with 0.5% Triton X-100 (Merck; X100) in PBS. After 1 h block with 1% bovine serum albumin (BSA, Merck; A3294) at room temperature, coverslips were incubated in a humidified chamber for 2 h at room temperature with an anti-GAC antibody (1:500, Gene Tex; GTX131263) and an anti-VDAC1 antibody (1:100 Santa Cruz Biotechnology, sc-390996). Afterward, coverslips were washed with PBS 3 times (5 min/wash) and incubated for 1 h with goat anti-rabbit IgG Alexa Fluor 555 or goat anti-mouse IgG Alexa Fluor 488 fluorescent secondary antibody (1:200, Invitrogen, Carlsbad, CA, USA). Finally, samples were washed with PBS 3 times (5 min/wash) and coverslips were mounted in ProLong Diamond Antifade Mountant (Life Technologies, Thermo Fisher Scientific, Carlsbad, CA, USA) and analyzed with an LSM510 confocal microscopy (Zeiss, Oberkochen, Germany).
3
Western Blot Analysis of OXPHOS Proteins
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Western blot analyses were performed in Bioplex‐lysates of human muscle tissue as previously described (Wefers et al., 2020 (link)). Equal amounts of proteins were loaded on gradient Bolt 4%–12% gels (Novex, Thermo Fisher Scientific). Proteins were transferred to nitrocellulose with the Trans‐Blot Turbo transfer system (Bio‐Rad Laboratories). The following antibodies and dilutions were used in this study: a cocktail of mouse monoclonal antibodies directed against human OXPHOS (dilution 1:5.000; ab110411, Abcam), as well as antibodies directed against TOMM20 (dilution 1:10.000; aba186734; Abcam), porin/VDAC (dilution 1:1.000; sc‐390,996; 1:5000, Santa Cruz biotechnology). The specific proteins were detected using secondary antibodies conjugated with IRDye680 or IRDye800 and were quantified with the CLx Odyssey Near Infrared Imager (Li‐COR, Westburg).
4
Quantification of Mitochondrial NOX4 Levels
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In order to assess NOX4 expression, 10% SDS-page was performed to resolve mitochondrial samples of 60 μg protein. Then, proteins were transferred to membranes of nitrocellulose and blocked with 7.5% non-fat dry milk in PBS-Tween for 1 h. Overnight incubation was done with anti-NOX4 (1:250; sc-30141, Santa Cruz Biotechnology) or anti-VDAC (1:500; sc-390996, Santa Cruz Biotechnology). PBS-Tween was used to wash the membranes for 10 min three times, and HRPconjugated anti-rabbit antibody (1:12500, 170-6515, BIO-RAD) or anti-mouse antibody (1:5000, 170-6516, BIO-RAD) were employed for secondary detection.
Membranes were washed as previously described and chemiluminescent detection was done using Clarity Max Western ECL substrate-Luminol Solution (BIO-RAD).
ImageJ program (1.50i, Wayne Rasband, National Institutes of Health, USA) was used to quantify bands (n=5 each group).
Membranes were washed as previously described and chemiluminescent detection was done using Clarity Max Western ECL substrate-Luminol Solution (BIO-RAD).
ImageJ program (1.50i, Wayne Rasband, National Institutes of Health, USA) was used to quantify bands (n=5 each group).
5
Immunohistochemical Analysis of Muscle Signaling
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Fresh gastrocnemius muscles were xed in 4% paraformaldehyde for 24 h and embedded in para n. Para n sections were cut in section at 4 μm thickness, then depara nized and rehydrated before antigen retrieval. Sections were blocked with 10% bovine serumal bumin (BSA) in TBS-Tween 20 (Sigma Aldrich) for 1 h at room temperature. Sections was incubated overnight at 4 °C with the respective following primary antibodies against PGC-1α (ab191838, Abcam, UK), NRF-1 (A14190, Abclonal, USA), NRF-2 (A8419, Abclonal, USA), VDAC1 (sc-390996, Santa Cruz, USA), AKT1 (10176-2-AP, Proteintech, USA) and GSK-3β (22104-1-AP, Proteintech, USA), and then incubated with secondary antibodies labelled with either Alexa Fluor 488 nm, FITC/Texas Red or biotin for 30 min at 37 °C. For IHC, slides were incubated with diaminobenzidine (DAB) for 5 min, and then counterstained with Gill's hematoxylin for 30 s. For IF, slides were incubated with Hoechst 33342 (1 μg/ml) for 5 min, then coverslipped with anti-fade media. Finally, images were captured on a uorescent microscope (ECLIPSE Ci-L, Nikon, Japan).
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