Islets isolated from 6-week Akita or WT B/6J mice were treated with STF 20 μM or DMSO vehicle (0.1%) for 3 h. Proteins were extracted with RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP40, 2 mM EDTA) plus protease inhibitor/phosphatase inhibitor cocktail (Sigma-Aldrich) and centrifuged at 4°C for 10 min at 10,000 g. Total protein concentration in the cell lysate was determined by BCA. Samples of ~ 20 μg protein prepared in Laemmli sample buffer without (non-reducing) or with (reducing) 5% b-mercaptoethanol were resolved on 4-12% Bis-Tris NuPAGE gels (Invitrogen) at 100 V for 60 min. The nonreducing gels were incubated in 25 mM dithiothreitol (DTT) solution for 10 min at room temperature before being transferred to PVDF membranes for 25 min at 4°C at 40 V. Membranes were probed with anti-proinsulin antibody (CCI-17, NOVUS) and HRP-conjugated secondary antibodies (1:3000; Santa Cruz Biotechnology, CA, USA).
Protease inhibitor phosphatase inhibitor cocktail
Manufactured by Merck Group
The Protease inhibitor/phosphatase inhibitor cocktail is a mixture of chemical compounds designed to inhibit the activity of proteases and phosphatases. This product is commonly used in biological research applications to prevent the degradation of proteins during sample preparation and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Nupage bis tris gel, by Thermo Fisher Scientific (3 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (2 mentions)
Hrp conjugated secondary antibody, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose, by Bio-Rad (1 mentions)
Peroxidase conjugated goat anti mouse igg, by Jackson ImmunoResearch (1 mentions)
Peroxidase conjugated goat anti rabbit igg, by Jackson ImmunoResearch (1 mentions)
Supersignal west pico plus, by Thermo Fisher Scientific (1 mentions)
3 protocols using protease inhibitor phosphatase inhibitor cocktail
1
Proinsulin Expression in Akita Mice
2
Protein Extraction and Analysis Protocol
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After removal of media, cells were washed once with ice-cold PBS and lysed in RIPA buffer (10 mM Tris pH 7.4, 150 mM NaCl, 0.1% SDS, 1% NP40, 2 mM EDTA) plus protease inhibitor/phosphatase inhibitor cocktail (Sigma-Aldrich) or directly in Laemmli gel sample buffer. Lysates in RIPA buffer were immediately spun at 10,000 rpm for 10 min at 4 °C and the supernatants analyzed further or stored at −80°C. Islets that had been quick frozen were placed on ice and RIPA buffer containing protease inhibitor cocktail and phosphatase inhibitor was added. Lysis was carried out by gently pipetting or by syringe through a 30G needle. Total protein concentration in the lysate was determined by BCA or Bramhall assay, and 5–10 μg of samples prepared in SDS sample were resolved by SDS-PAGE in 4–12% Bis-Tris NuPAGE gels (Invitrogen) at 200 V for 30 min. Nonreducing gels were incubated in a solution containing 25 mM dithiothreitol (DTT) for 10 min at room temperature prior to electrotransfer.
3
Analyzing Protein Expression in Mouse β-Cells
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Min6 mouse β-cells (25 (link)) (obtained from Dr. D. Stoffers, University of Pennsylvania, Philadelphia, PA) were cultured in DMEM supplemented with 10% FBS, penicillin/streptomycin, and 0.05 mmol/L β-mercaptoethanol. Cells at 70–80% confluency were transfected using Lipofectamine 2000 (Thermo Fisher Scientific), with fresh media changed at 6 h posttransfection. Media were removed at 48 h; the cells were washed with ice-cold PBS and lysed in radioimmunoprecipitation assay buffer (10 mmol/L Tris, pH 7.4, 150 mmol/L NaCl, 0.1% SDS, 1% NP40, 2 mmol/L EDTA) plus protease inhibitor/phosphatase inhibitor cocktail (Sigma-Aldrich). Total protein was measured by bicinchoninic acid assay. Proteins in sample buffer were boiled and resolved by 4–12% NuPAGE Bis-Tris Gel (Invitrogen) and electrotransferred to nitrocellulose (Bio-Rad). Membranes were incubated with primary antibodies (4°C overnight) and then secondary peroxidase-conjugated goat anti-rabbit IgG (111-035-144; Jackson ImmunoResearch) or peroxidase-conjugated goat anti-mouse IgG (115-035-174; Jackson ImmunoResearch) followed by enhanced chemiluminescence (SuperSignal West Pico PLUS; Thermo Fisher Scientific) with digital image capture.
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