BAT, iWAT, and gWAT depots were homogenized with 300–500 ul of RIPA buffer (50 mM Tris–HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.25% deoxycholate) containing protease inhibitor cocktail (Roche) and phospho-STOP cocktail (Roche), followed by pelleting of the insoluble debris at 13,000×g for 15 min at 4 °C. The protein concentrations of lysates were determined by BCA assay (Thermo Scientific), and 30 μg of lysate was separated by Tris-glycine SDS-PAGE (10% polyacrylamide). Proteins were transferred to nitrocellulose membranes (Protran BA 83, Whatman), blocked in 3% BSA in 1X TBST (Tris-buffered saline with Tween 20), and incubated with primary antibodies overnight. The blots were probed with the following antibodies: Ucp1 (Sigma; U6382), Ndufb8, Sdhb, Uqcrc2, Atp5a (MitoProfile total OXPHOS, Abcam; ab110413), Aco2 (Cell Signaling; 6922), Mcad (GeneTex; GTX32421), Pcx (Abcam; ab128952), Vdac (Calbiochem; PC548), Pdh E2/E3bp (Abcam; ab110333), Acot2 (Sigma; SAB2100030), beta-Actin (Sigma; A2228), and Hsc-70 (Santa Cruz; sc-7298). Cy3-conjugated anti-mouse (Invitrogen), or HRP-conjugated anti-mouse (GE Healthcare) or anti-rabbit (GE Healthcare) secondary antibodies were used appropriately. Images were collected and analyzed using an Alpha Innotech FluorChemQ.
Phospho stop cocktail
Manufactured by Roche
Phospho-STOP cocktail is a solution designed to inhibit phosphatase enzymes, which are responsible for dephosphorylating proteins. This product can be used to preserve the phosphorylation state of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Fluorchem q, by Bio-Techne (1 mentions)
Bca assay, by Thermo Fisher Scientific (1 mentions)
Protran ba83, by Cytiva (1 mentions)
Ab110413, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Roche (1 mentions)
U6382, by Merck Group (1 mentions)
Whatman, by Cytiva (1 mentions)
Protran, by Cytiva (1 mentions)
Pvdf membrane, by Merck Group (1 mentions)
Sodium dodecyl sulfate (sds), by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
2 protocols using phospho stop cocktail
1
Western Blot Analysis of Adipose Tissue
2
Protein Extraction and Western Blot Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
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Cells were lysed in ice-cold RIPA buffer (50mM Tris-HCl, pH 7.4, 150 mM NaCl, 1% NP-40, 0.5% sodium deoxycholate, 0.1% SDS (Sigma)) supplemented with proteinase inhibitors (2 μg/ml leupeptin, 2 μg/ml aprotinin, 1 mM PMSF (Sigma)) and Phospho-stop cocktail (Roche). Cell lysates were sonicated at 80% amplitude for 10 seconds and then cleared by centrifugation at 13000 rpm for 15 minutes. Total protein content was measured for each sample using the BCA assay (Pierce) before 5× loading buffer (bromophenol blue (0.25%), glycerol (50%), SDS (sodium dodecyl sulfate; 10%), Tris-Cl (0.25 M, pH 6.8), mercaptoethanol (3.6%)) was added and protein samples were boiled at 95°C for 10 minutes. Equal amounts of protein were loaded and separated on 8−15% SDS-PAGE gels. Proteins were then transferred onto PVDF membranes (Millipore) using wet transfer with transfer buffer (25 mM Tris base, 189 mM glycine, 20% methanol) at 4°C. Membranes were washed in PBST and blocked with 5% non-fat milk in TBST for 1 hour, followed by sequential incubation with primary and secondary antibodies. Signals were detected using an Odyssey Clx membrane scanner.
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