Immediately after collection, the hypothalamus and mammary glands were homogenized in RIPA buffer (Sigma) containing a cocktail of protease and phosphatase inhibitors (1:100, Sigma) and centrifuged (14000 RPM, 4 °C for 20 minutes), and the supernatants were retained. After determining the total protein concentration (Pierce BCA Protein Assay, Thermo Scientific), 50 μg of total protein was loaded on a 10% SDS-PAGE gel, and the separated proteins were then transferred to a nitrocellulose membrane (Bio-Rad). Membranes were blocked with 5% bovine serum albumin and incubated overnight at 4 °C using commercially available primary antibodies (1:1000) to identify pSTAT3Tyr705 (Cell Signaling), pSTAT5Tyr694 (Cell Signaling), GAPDH (Santa Cruz) or α-tubulin (Cell Signaling). Next, we incubated the membranes for 45 min in 1:10,000 secondary antibody (IRDye 800CW, Li-COR). Proteins were detected by fluorescence and analyzed using the Li-COR Odyssey system (Li-COR), and protein amounts were normalized to GAPDH or α-tubulin amounts.
Pierce bca protein assay
Manufactured by Bio-Rad
Sourced in United States
The Pierce BCA Protein Assay is a colorimetric assay used for the quantitative determination of total protein concentration in a sample. It utilizes the bicinchoninic acid (BCA) reaction, where proteins reduce Cu2+ to Cu+ in an alkaline environment, and the Cu+ ions then chelate with BCA to produce a purple-colored complex that absorbs light at 562 nm. This assay provides a simple, sensitive, and accurate method for measuring total protein levels in a wide variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Cocktail of protease and phosphatase inhibitors, by Merck Group (1 mentions)
Odyssey system, by LI COR (1 mentions)
P stat5 tyr694, by Cell Signaling Technology (1 mentions)
α tubulin, by LI COR (1 mentions)
Gapdh, by LI COR (1 mentions)
α tubulin, by Cell Signaling Technology (1 mentions)
Irdye 800cw, by LI COR (1 mentions)
P stat3 tyr705, by Cell Signaling Technology (1 mentions)
Ripa buffer, by Merck Group (1 mentions)
Gapdh, by Santa Cruz Biotechnology (1 mentions)
Nitrocellulose membrane, by Bio-Rad (1 mentions)
Protease inhibitor, by Promega (1 mentions)
Mouse anti tubulin, by Santa Cruz Biotechnology (1 mentions)
Rabbit anti cleaved notch1, by Cell Signaling Technology (1 mentions)
Sc 8035, by Cell Signaling Technology (1 mentions)
Polyvinylidene difluoride membrane, by Bio-Rad (1 mentions)
Skim milk, by Nacalai Tesque (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Phosphatase inhibitor, by Thermo Fisher Scientific (1 mentions)
Radioimmunoprecipitation assay buffer, by Nacalai Tesque (1 mentions)
3 protocols using pierce bca protein assay
1
Western Blot Analysis of Phosphorylated STAT3/5
2
Quantifying Notch1 Activation in Myogenic Progenitors
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Sorted myogenic progenitors from PT, PTC, and CTL iPSCs from five biological replicates were collected and quantified for N1ICD protein expression using WB. Briefly, cells were harvested in RIPA buffer supplemented with protease inhibitors (Promega). The Pierce BCA Protein Assay (Bio-Rad) was used to quantify total protein levels, using bovine serum albumin as a standard. Protein extracts (25 μg per well) were separated on a 4%–15% SDS-PAGE Mini-PROTEAN TGX Precast Gradient Gel (Bio-Rad) and transferred onto polyvinylidene difluoride membranes (Bio-Rad). Membranes were blocked in 5% skim milk powder dissolved in 0.1% TBS-T at room temperature for 1 h. The membrane was incubated with primary antibodies (mouse anti-tubulin, Santa Cruz Biotechnology, sc-8035, 1:1,000; rabbit anti-cleaved NOTCH1, Cell Signaling Technology, 4147, 1:1,000) overnight at 4°C. The following secondary antibodies were used: horseradish peroxidase (HRP)-linked anti-mouse (Jackson ImmunoResearch, 115-035-003, 1:10,000) and anti-rabbit (Jackson ImmunoResearch, 111-035-003, 1:10,000), followed by enhanced chemiluminescence (ECL) detection. Captured blot images were used for band intensity quantification using ImageJ software.
3
Western Blot Analysis of Protein Expression
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HEKa cells were washed with Tris‐buffered saline (TBS, Nacalai Tesque) and lysed in radioimmunoprecipitation assay buffer (Nacalai Tesque) containing protease inhibitors (Millipore) and phosphatase inhibitors (Thermo Fisher Scientific). Equal amounts of total protein quantified using the Pierce BCA Protein Assay were separated using sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to a polyvinylidene fluoride membrane (Bio‐Rad Laboratories, CA, USA). After blocking for 1 h with 5% skim milk (Nacalai Tesque) or 0.5% bovine serum albumin (BSA) solution (Nacalai Tesque), the membranes were incubated with the antibodies listed in Table 1 , with β‐actin as a loading control. Signals were detected using SuperSignal™ West Dura Extended Duration Substrate (Thermo Fisher Scientific) with a Lumino Imager (LAS‐4000 mini, Fujifilm).
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