Anti-RhoA antibody [1B12] (ab54835)

Protocol tips

Upstream tips	Protocol tips	Downstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.	-Mouse (1:100) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Upstream tips
-These buffers may be stored at 4°C for several weeks or aliquoted and stored at -20°C for up to a year.
Protocol tips
-Mouse (1:100) -To reduce and denature your samples, boil each cell lysate in sample buffer at 100°C for 5 min. Lysates can be aliquoted and stored at -20°C for future use.

Publication protocol

Two days post-confluence, endothelial cell monolayers on 2.5, 5, and 10 kPa PA gels and glass controls were treated with 0 or 10 μM simvastatin for 24 hours. Samples for phosphorylated myosin light chain (pMLC) analysis were lysed directly into boiling 2x Laemmli buffer, followed by immediate heating of the lysate at 95°C and heavy vortexing to disrupt nucleic acid structures. Lysate collected for the RhoA and Rac1 assays were used for quantifying total GTPase protein expression. Lysate was separated using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) followed by protein transfer onto a polyvinyl difluoride (PVDF) membrane (Bio-Rad). Total RhoA was probed with a mouse monoclonal antibody against full-length RhoA (1:100) (Abcam, No. ab54835). Phosphorylated myosin light chain (pMLC) was probed with a polyclonal rabbit antibody against pMLC at threonine-18 and serine-19 (1:50) (Cell Signaling Technology, No. 3674). Total Rac1 was probed with a mouse monoclonal antibody against full-length Rac1 (1:00) (Millipore, No. 23A8). Alpha tubulin was probed as a loading control with a mouse polyclonal primary antibody (1:2000) (Sigma, No. T3559). Horseradish peroxidase (HRP) conjugated anti-rabbit and anti-mouse secondary antibodies were used (1:2000) (Rockland, No. 611-103-122 and Rockland, No. 610-103-121, respectively). The signal was developed with SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific). The membranes were exposed and imaged with a FujiFilm Image-Quant LAS-4000, followed by protein quantification using ImageJ software (v2.0.0-rc-41/1.50d, National Institutes of Health, Bethesda, MD, USA). The pMLC, RhoA, and Rac1 signals were normalized to the alpha tubulin loading control at each condition.

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Manufacturer protocol

Download the product protocol from Abcam for Anti-RhoA antibody [1B12] (ab54835) below.

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