Whole cell lysates were prepared using boiling lysis buffer (1% SDS, 10 mM Tris.HCl, pH 74.). Protein concentrations were determined using BCA reagent (Thermo Scientific, Rockford, IL) and equal amounts of proteins were separated onto Criterion or mini gradient polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes (Bio-Rad). The following primary antibodies were used: rabbit antibodies for phospho-S6 (Ser235/236), phospho-S6K(T389 ) and total S6K - were from Cell Signaling Biotechnology. Mouse anti-S6 antibody was from Cell Signaling Biotechnology. Anti-β-actin-HRP antibody was from Sigma-Aldrich (St. Louis, MO); mouse antibody for p53 (Ab-6) was from Oncogene Research products (La Jolla, CA). Secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were from Cell Signaling Biotechnology as previously described [20 (link)] , [21 (link)] .
Mini gradient polyacrylamide gels
Manufactured by Bio-Rad
Sourced in United States
Mini gradient polyacrylamide gels are laboratory equipment used for electrophoresis separation of biomolecules such as proteins and nucleic acids. They feature a linear gradient of polyacrylamide concentrations across the gel, enabling simultaneous separation of molecules with a wide range of molecular weights.
Automatically generated - may contain errors
Lab products found in correlation
Anti rabbit and anti mouse hrp conjugated secondary antibodies, by Cell Signaling Technology (3 mentions)
Mouse anti s6 antibody, by Cell Signaling Technology (2 mentions)
Anti β actin hrp antibody, by Merck Group (1 mentions)
Bca reagent, by Thermo Fisher Scientific (1 mentions)
Pvdf membrane, by Bio-Rad (1 mentions)
Phospho erk, by Cell Signaling Technology (1 mentions)
Phospho akt s473, by Cell Signaling Technology (1 mentions)
Anti gapdh antibody, by Merck Group (1 mentions)
Phospho s6 s235 236, by Cell Signaling Technology (1 mentions)
Phospho thr 389 p70s6k, by Cell Signaling Technology (1 mentions)
Rabbit anti actin antibody, by Merck Group (1 mentions)
3 protocols using mini gradient polyacrylamide gels
1
Western Blot Analysis of Signaling Proteins
2
Western Blot Analysis of Phospho-proteins
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Immunoblotting was performed as described previously.31 (link) In brief, whole-cell lysates were prepared using boiling lysis buffer (1% SDS, 10 mM Tris·HCl, pH 7.4). Equal amounts of proteins were separated using Criterion or mini gradient polyacrylamide gels (Bio-Rad, Hercules, CA, USA) and transferred to PVDF membranes. The following rabbit antibodies for: phospho-S6 (S235/236), phospho-S6 (S240/244), phospho ERK½, phospho-Thr 389 p70S6K, phospho-AKT (S473) and phospho-AKT (T308), phospho-IRS1 (S1101), phospho-IRS1(S636/639), IRS1 and mouse anti-S6 antibody were from Cell Signaling Biotechnology (Danvers, MA, USA). Rabbit anti-actin and mouse monoclonal anti-GAPDH antibodies were from Sigma-Aldrich and Invitrogen (Camarillo, CA, USA), respectively. Secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were from Cell Signaling Biotechnology.
3
Western Blot Analysis of Signaling Pathways
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Whole cell lysates were prepared using boiling lysis buffer (1% SDS, 10 mM Tris.HCl, pH 74.). Equal amounts of proteins were separated using Criterion or mini gradient polyacrylamide gels (Bio-Rad, Hercules, CA) and transferred to PVDF membranes. The following rabbit antibodies for: phospho-S6 (Ser235/236 and S240/244), phospho-AKT (T308), phospho ERK ½, AKT, phospho-4EBP1(T37/46) and phospho-S6K(T421/S424)- were from Cell Signaling Biotechnology (Danvers, MA). Mouse anti-phospho-Thr 389 -S6K and anti-S6 antibody were from Cell Signaling Biotechnology. Rabbit anti-actin antibody was from Sigma-Aldrich (St. Louis, MO); mouse antibodies for p21 and cyclin D1 were from from BD Biosciences (San Jose, CA) and Santa Cruz Biotechnology (Paso Robles, CA), respectively. Secondary anti-rabbit and anti-mouse HRP-conjugated antibodies were from Cell Signaling Biotechnology.
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