Total cell lysates were prepared with the lysis buffer (20 mM Tris, pH 7.4, 150 mM NaCl, 2 mM EDTA, and 1% Triton X-100), and proteins were separated with sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). Then, separated proteins were electrophoretically transferred from the gel to a polyvinylidene fluoride membrane (Millipore, MA, USA). Membranes were blocked and incubated overnight with primary antibodies diluted in PBS (pH 7.4), 0.2% Tween-20, 5% bovine serum albumin and 0.002% sodium azide. Primary antibodies used for western analyses were RhoA (1:1000, Cell Signaling) and tubulin (1:2000, Sigma). Then, they were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (1:2000, Cell Signaling) and developed with Immobilon western chemiluminescence substrate (Millipore) in a Bio-Rad ChemiDoc EQ system.
Immobilon western chemiluminescence substrate
Manufactured by Merck Group
Immobilon western chemiluminescence substrate is a laboratory reagent designed for the detection of proteins in Western blot analysis. It is a chemiluminescent substrate that reacts with the enzyme-labeled secondary antibody, producing a luminescent signal proportional to the amount of target protein present. This substrate is used to visualize and quantify proteins separated by gel electrophoresis and transferred to a membrane.
Automatically generated - may contain errors
Lab products found in correlation
Horseradish peroxidase conjugated secondary antibody, by Cell Signaling Technology (1 mentions)
Polyvinylidene fluoride membrane, by Merck Group (1 mentions)
Chemidoc eq system, by Bio-Rad (1 mentions)
Amersham imager 600, by GE Healthcare (1 mentions)
Bradford assay kit, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Amersham imager, by Cytiva (1 mentions)
2 protocols using immobilon western chemiluminescence substrate
1
Western Blot Analysis of RhoA
2
Cell Lysis and Protein Quantification
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2 × 105 cells/mL were plated in 6-well plates and treated with compounds as indicated. Cells were collected, washed with cold 1 × PBS, and lysed in 1 × SDS buffer containing protease inhibitor cocktails (cat.539134, Merck). Protein in cell lysate was quantified by detergent-compatible Bradford assay kit (cat.23246, Thermo). The Millipore immobilon Western chemiluminescence substrate was used for signal development. Blots were imaged in an Amersham Imager 600 (G.E. Healthcare).
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