Total cell extracts were prepared using lysis buffer (20 mM HEPES [pH 7.4], 1% Triton X-100, 1 mM EDTA, 1 mM MgCl2, 150 mM NaCl, 10% glycerol, protease inhibitor, and phosphatase inhibitor cocktail [Invitrogen]). Protein concentrations were determined using micro-BCA protein reagent (Pierce Biotechnology). Thirty micrograms of total proteins were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes with 0.2-μm pore size (Whatman). The membranes were incubated with antibodies against phospho-AKT (Ser473) (#4060, 1:1000; Cell Signaling Technology (CST); RRID: AB_2315049), AKT (#9272, 1:1000; CST; RRID: AB_329827), phospho-mTOR (Ser2448) (#2971, 1:1000; CST; RRID: AB_330970), mTOR (#2972, 1:1000; CST; RRID: AB_330978), phospho-S6 ribosomal protein (Ser2235/236) (#2211, 1:1000; CST; RRID: AB_331679), S6 ribosomal protein (#2217, 1:1000; CST; RRID: AB_331355), phospho-4E-BP1 (Thr70) (#13396, 1:1000; CST; RRID: AB_2798206), 4E-BP1 (#9644, 1:1000; CST; RRID: AB_2097841), phospho-EGFR (Tyr1068) (#3777, 1:1000; CST; RRID: AB_2096270), EGFR (#2646, 1:1000; CST; RRID: AB_2230881), RNF11 (ab154831, 1:1000; Abcam), or β-actin (AC-15, 1:5000; Sigma; RRID: AB_476692). The ECL method (Invitrogen) was used for protein detection.
Micro bca protein reagent
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Micro-BCA protein reagent is a colorimetric detection kit used for the quantitative determination of protein concentration in small-volume samples. It is based on the bicinchoninic acid (BCA) method, which provides a sensitive and robust assay for measuring total protein. The reagent allows for the accurate measurement of protein levels in a wide range of sample types, including cell lysates, purified proteins, and other biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Phosphatase inhibitor cocktail, by Thermo Fisher Scientific (2 mentions)
Nitrocellulose membrane, by Cytiva (2 mentions)
Ecl system, by Thermo Fisher Scientific (2 mentions)
β actin, by Merck Group (2 mentions)
Protease inhibitor, by Thermo Fisher Scientific (1 mentions)
Ecl method, by Thermo Fisher Scientific (1 mentions)
N cadherin, by Cell Signaling Technology (1 mentions)
Angiopoietin 2, by AbFrontier (1 mentions)
Occludin, by Cell Signaling Technology (1 mentions)
Protease inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Twist, by Santa Cruz Biotechnology (1 mentions)
Snail, by Cell Signaling Technology (1 mentions)
Proliferating cell nuclear antigen (pcna), by Santa Cruz Biotechnology (1 mentions)
Vimentin, by Cell Signaling Technology (1 mentions)
3 protocols using micro bca protein reagent
1
Protein Extraction and Western Blot Analysis
2
Protein Expression Analysis in Cell Extracts
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Total cell extracts were obtained using lysis buffer (20 mM HEPES [pH 7.4], 1% Triton X‐100, 1 mM EDTA, 1 mM MgCl2, 150 mM NaCl, 10% glycerol, protease inhibitor, and phosphatase inhibitor cocktail [Invitrogen]) and the protein concentration was determined using the micro‐BCA protein reagent (Pierce Biotechnology). Equal amounts of protein (30 μg) from the clarified lysates were separated by SDS‐PAGE and transferred onto nitrocellulose membranes having a 0.2 μm pore size (Whatman). The membranes were incubated in antibodies against N‐cadherin (#13116, 1:1000; Cell Signaling Technology [CST]), E‐cadherin (#14472, 1:1000; CST), vimentin (#5741, 1:1000; CST), SNAIL (#3879, 1:1000; CST), VEGFR2 (#9698, 1:1000; CST), Smad2/3 (#3102, 1:1000; CST), phosphor‐Smad2 (Ser 465/467)/Smad3 (Ser 423/425) (#8828, 1:1000; CST), β‐catenin (#8480, 1:1000; CST), cytokeratin (ab9023, 1:2000; Abcam), and β‐actin (AC‐15, 1:5000; Sigma) An ECL system was used for protein detection (Invitrogen).
3
Western Blot Analysis of Cell Signaling
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Total cell extracts were obtained using lysis buffer (20 mM HEPES [pH 7.4], 1% Triton X-100, 1 mM EDTA, 1 mM MgCl2, 150 mM NaCl, 10% glycerol, and protease inhibitor cocktail [Invitrogen]), and protein concentration was determined using the micro-BCA protein reagent (Pierce Biotechnology, Rockford, IL, USA). Equal amounts of proteins (30 μg per well) from clarified lysates were separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis and transferred onto nitrocellulose membranes with 0.45-μm pores (Whatman, Maidstone, UK). The membranes were sequentially incubated in 5% dry milk and antibodies against RRAD (Abcam, Cambridge, U.K, ab75100, 1:1000), β-actin (Sigma, St Louis, MO, USA, A5441, 1:5,000), PCNA (Santa Cruz Biotechnology, CA, sc-56, 1:1,000), vimentin (Cell Signaling, Danvers, MA, 5741S, 1:1,000), Twist (Santa Cruz, sc-15393, 1:1,000), snail (Cell Signaling, 3879S, 1:1,000), Occludin (Cell Signaling, 5446S, 1:1000), angiopoietin 2 (AbFrontier, Seoul, Korea, LF-PA50005, 1:500), and BiP (BD Biosciences, San Jose, CA, USA, 610978, 1:1,000). The ECL system was used for protein detection (Invitrogen).
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