Western blotting Smooth muscle actin

Western blotting is a widely used technique to size separate proteins from a pool of cell or tissue lysates. The technique has 4 major steps: a) gel electrophoresis, b) blocking and treatment with antigen specific antibody, c) treatment with secondary antibody and finally d) detection and visualization. Though western blotting is a widely used technique, detection of specific proteins depends on several factors, the major ones are antibody concentration, incubation time and washing steps. Key points for obtaining clean blots are: always prepare fresh buffer solutions and optimize antibody concentration. Given the advent of high-throughput protein analysis and a push to limit the use of lab consumables, onestep antibodies are developed which recognise protein of interest and also contain a detection label.

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Found 3 matching solutions for this experiment

Recombinant Anti-alpha smooth muscle Actin antibody [E184] (ab32575)

Abcam

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.	-Rabbit (1:5000) -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
-Rabbit (1:5000) -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.

Manufacturer protocol Publication protocol 1 Relevant paper

alpha-Smooth Muscle Actin Antibody

Novus Biologicals

Upstream tips	Protocol tips	Downstream tips
	-Goat (1:100) --Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Protocol tips
-Goat (1:100) --Centrifuge cell lysate mixture at 4˚C. The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. -Do not let the membrane dry at any point during the blotting process.

Manufacturer protocol Publication protocol 1 Relevant paper

Monoclonal Anti-ACTA2 antibody produced in mouse

Sigma-Aldrich

Upstream tips	Protocol tips	Downstream tips
	-Mouse (1:10,000)

Protocol tips
-Mouse (1:10,000)

Publication protocol 1 Relevant paper

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