To monitor protein levels, protein extracts were prepared in RIPA buffer (125 mM NaCl, 25 mM Tris-Cl pH7.4, 1 mM EGTA-Tris pH 7.4, 1% Triton X-100, 0.5% sodium deoxycholate, 0.1% SDS and Complete EDTA-free protease inhibitor mixture-Roche-). 40 μg of total proteins were loaded, according to BCA quantification. Proteins were separated by SDS-PAGE electrophoresis, in commercial 4%–12% acrylamide gels (Thermo Fisher Scientific) and transferred onto nitrocellulose membranes (Thermo Fisher Scientific) by wet electrophoretic transfer. Blots were blocked 1 h at RT with 5% non-fat dry milk (Bio-Rad) in TBS-tween (0.5M Tris, 1.5M NaCl, 0.01% Tween) solution and incubated at 4°C with primary antibodies. Secondary antibodies were incubated 1 h at RT. The following antibodies were used: anti-MCU (1:1000, Sigma-Aldrich), anti-MICU1 (1:1000 Novus Biologicals), anti-MICU2 (1:1000 Sigma-Aldrich), anti-GRP75 (1:5000, Santa Cruz Biotechnology) and anti-TOM20 (1:20000, Santa Cruz Biotechnology). Secondary HRP-conjugated antibodies were purchased from Bio-Rad and used at 1:5000 dilution.
Anti micu2
Manufactured by Merck Group
Anti-MICU2 is a laboratory equipment product that serves as a tool for researchers and scientists. It is designed to detect and measure the levels of MICU2, a protein involved in the regulation of mitochondrial calcium uptake. The core function of Anti-MICU2 is to provide researchers with a reliable method to analyze and quantify MICU2 expression in various biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti grp75, by Santa Cruz Biotechnology (2 mentions)
Acrylamide gel, by Thermo Fisher Scientific (1 mentions)
Anti mcu, by Merck Group (1 mentions)
Anti tom20, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions)
Non fat dry milk, by Bio-Rad (1 mentions)
Anti mfn2, by Abcam (1 mentions)
Anti oxphos, by Abcam (1 mentions)
Anti ip3r1, by Santa Cruz Biotechnology (1 mentions)
Anti pdh e1α, by Abcam (1 mentions)
Anti hsp60, by Abcam (1 mentions)
Anti vdac, by Abcam (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Anti gapdh, by Abcam (1 mentions)
2 protocols using anti micu2
1
Mitochondrial Protein Quantification by Western Blot
2
Immunoblotting Analysis of Mitochondrial Proteins
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Twenty to thirty micrograms of total or mitochondrial protein was separated on SDS-polyacrylamide gel and electro-blotted onto a nitrocellulose membrane (Bio-Rad). Membranes were saturated with blocking buffer for 1 h at room temperature and incubated overnight at 4°C with monoclonal mouse anti-VDAC (1:1000, Abcam), anti-OXPHOS (1:5000, Abcam), anti-IP3R1 (1:1000, Santa Cruz), anti-NDUFA13 (1:1000, Abcam), anti-GAPDH (1:10 000, Abcam) or with polyclonal rabbit anti-MCU (1:500, Abcam), anti-MICU1 (1:500, Thermo Fisher), anti-MICU2 (1:500, Sigma Aldrich), anti-PDH-E1α (1:1000, Abcam), anti-PDHE1α phosphor Ser 293 (1:1000, Abcam), anti-PDHE1α phosphor Ser 300 (1:1000, Millipore), anti-PDHE1α phosphor Ser 232 (1:1000, Calbiochem), anti-PDK4 (1:1000 Novus Biotech), anti-GRP75 (1:1000, Santa Cruz), anti-SIGMA1R (1:1000, Cell Signaling), anti-MFN2 (1:1000, Abcam), and anti-Hsp60 (1:1000, Abcam). Hsp60 and GAPDH were used as loading controls. All immunoblots were developed and quantified using the Odyssey infrared imaging system (LICOR Biosystems) and infrared-labeled secondary antibodies. Band intensities were quantified with ImageJ.
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