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

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.	-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.

Publication protocol

The cells were digested by 0.25% trypsin-EDTA and total proteins were extracted by cell lysis buffer supplemented with protease inhibitors. Equal amount of total proteins from each group was separated by SDS/PAGE and the proteins needed were transferred onto PVDF membranes. The membranes were incubated with 5% skimmed milk for 2 h, followed by primary antibodies of Wnt3a (1:2000), GSK-3β (1:1000), phospho-GSK-3β (Ser9) (1:1000), β-catenin (1:1000), and α-SMA (1:5000) overnight at 4°C. Subsequently, the membranes were washed and incubated with goat anti-rabbit (1:100000) or mouse (1:5000) horseradish peroxidase-conjugated secondary antibody for 1 h. Finally, the membranes were washed again and ECL detection reagent was added to make protein bands to be seen under ImageQuant LAS4000 chemiluminescence detection system (GE Healthcare, U.K.). The protein levels were quantified by ImageJ software and the gray values were normalized to GAPDH. The data were obtained from at least three independent experiments.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-alpha smooth muscle Actin antibody [E184] (ab32575) below.

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