Cell and tissue lysates were prepared with 1% SDS buffer. The protein concentrations were measured by using a DC Protein Assay Kit (Bio-Rad). Protein samples (1–5 μg/lane) were resolved in 10%–12.5% SDS-PAGE gells and transferred to 0.45 μm PVDF membranes (Immobilon-P Membrane, EMD Millipore). After having been blocked with PVDF Blocking Reagent for Can Get Signal (TOYOBO) for 1 hour, the membranes were incubated overnight at 4°C with primary antibodies. The primary antibodies used for immunoblotting are summarized in Supplemental Table 2 . The membranes were incubated at room temperature with HRP-linked anti–mouse IgG or anti–rabbit IgG as secondary antibodies (Cell Signaling Technology) diluted 1:4000. After 3 washes with TBS-T, the immunoblots were visualized by using the Luminata Forte Western HRP substrate (EMD Millipore). The loaded amount was verified with an anti–β-actin mouse monoclonal antibody (clone AC-74, Sigma-Aldrich, A5316). Please see Supplemental Methods for detailed information.
Hrp linked anti mouse igg or anti rabbit igg
Manufactured by Cell Signaling Technology
Sourced in United States
HRP-linked anti-mouse IgG or anti-rabbit IgG is a secondary antibody conjugated with horseradish peroxidase (HRP). It is used to detect and quantify mouse or rabbit primary antibodies in various immunoassays.
Automatically generated - may contain errors
Lab products found in correlation
Immobilon p membrane, by Merck Group (2 mentions)
Pvdf blocking reagent for can get signal, by Toyobo (1 mentions)
Luminata forte western hrp substrate, by Merck Group (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
Mouse anti gapdh, by Merck Group (1 mentions)
Halt protease and phosphate inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Ecl plus, by GE Healthcare (1 mentions)
Bovine serum albumin (bsa), by Merck Group (1 mentions)
Protein assay, by Bio-Rad (1 mentions)
Alpha tubulin, by New England Biolabs (1 mentions)
Clarify western ecl substrate, by Bio-Rad (1 mentions)
Restore western blot stripping buffer, by Thermo Fisher Scientific (1 mentions)
Chemidoc xrs system, by Bio-Rad (1 mentions)
3 protocols using hrp linked anti mouse igg or anti rabbit igg
1
Western Blot Analysis of Protein Samples
2
Western Blot Analysis of H-Ras Signaling
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Cells were transiently transfected with EGFP-H-Ras plasmid and then lysed in radioimmunoprecipitation assay buffer added with DTT (0.5 mM), and protease and phosphatase inhibitors (100 × Halt protease and phosphate inhibitor cocktail, no. 1861281, Thermo Scientific, Waltham, MA, USA). The protein concentration of the cell lysates was determined by a protein assay (Bio-Rad Protein Assay, Bio-Rad, Hercules, CA, USA). The cell were subjected to SDS-PAGE, and then transferred to immobilon-P membrane (Millipore, Billerica, MA, USA). After blocking with 1.5% bovine serum albumin in TBS (blocking solution), the membrane was incubated overnight (4 °C) in primary antibody diluted (1 : 1000) in 1.5% bovine serum albumin (Sigma-Aldrich). Antibodies utilized in the current study are as follows: rabbit anti-phospho ERK(T202/Y204) (no. 9101), rabbit anti-total ERK (no. 9102), rabbit anti-HMGR (Millipore ABS#229), and mouse anti-GAPDH (no. MAB374, Millipore). The membrane was then washed in TBS, and incubated (1 h at RT) with HRP-linked anti-mouse IgG or anti-rabbit IgG diluted (1 : 5000) in 1.5% bovine serum albumin-TBS (no. 7074 and no. 7076, respectively, Cell Signaling, Beverly, MA, USA). The membrane was then washed in TBS, and analyzed with ECL Plus (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) by autoradiography film.
3
Western Blot Analysis of MAPKs
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Samples were diluted to 20 μg/mL in a 1:5 ratio with Laemelli sample buffer, heated for 10 minutes at 95°C, and subjected to SDS-PAGE on a 12% polyacrylamide gel, using All Blue Prestained Protein Standards (Bio-RAD, Watford, Hertfordshire, UK). Proteins were transferred to Hybond P 0.45 μm PVDF membrane (Fisher Scientific), blocked for 1 hour in blocking buffer (5% BSA, 20 mM Tris pH 7.6, 137 mM NaCl and 0.1% v/v Tween 20; all Sigma-Aldrich), and then incubated overnight at 4°C in blocking buffer with 1:1000 dilution of primary antibody for total and phosphorylated JNK, p38, and ERK (Supporting Information Table S2). The membrane was washed 5× in wash buffer (20 mM Tris pH7.6, 125 mM NaCl, 0.1% v/v Tween 20) and bound antibodies identified by incubation for 1 hour at room temperature with 1:1000 HRP-linked anti-mouse IgG or anti-rabbit IgG (Cell Signaling Technology Inc, Danvers, MA, USA), washed 5× in wash buffer, and visualized by chemiluminescence (Clarify Western ECL Substrate, Bio-RAD) and a BIO-RAD ChemiDoc XRS system. The membrane was stripped using Restore Western Blot Stripping Buffer (Thermo Fisher Scientific) to detect another MAPK, or alpha-tubulin (New England Biolabs, Hitchin, Hertfordshire, UK). The density of protein bands was analyzed by ImageJ.
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