Recombinant Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547)

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 (1:1000) --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 (1:1000) --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

Cells were harvested and lysed in RIRA cell lysate (50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS and 0.5 mM PMSF) (Beyotime Biotechnology, Beijing, China), and total proteins were extracted. The proteins were quantified by BCA assay kit (Beyotime Biotechnology, Beijing, China), and 5 × loading buffer (250 mM Tris-HCl (pH 6.8), 10% SDS, 0.5% BPB, 50% Glycerin, 5% 2-Mercaptoethanol) was added to the proteins. Next, 35 μg total proteins from each sample were uploaded and separated in 12% SDS-PAGE at 120 V voltage, then transferred onto PVDF membranes at 40 mA for 2.5 h at room temperature. According to the standard protein marker, the PVDF membrane was cut according to the molecular weight of the target protein. After blocking by 5% defatted milk powder for 2 h at room temperature, PVDF membrane bands were incubated with primary antibodies overnight at 4 °C, such as, c-Src (1:1000, ab109381, Abcam, Cambridge, UK), p-Src418 (1:500, ab133460, Abcam, Cambridge, UK), E-Cadherin (1:200, #3195, Cell Signal Technology, Danvers, MA, USA), ZO-1 (1:1000, 21,773–1-AP, Proteintech, Chicago, IL, USA), Vimentin (1:1000, ab92547, Abcam, Cambridge, UK), α-SMA (1:1000, ab124964, Abcam, Cambridge, UK) and GAPDH (1:2000, Cell Signal Technology, Danvers, MA, USA). After being washed with TBST three times (for 15 mins each) the next day, PVDF membrane bands were incubated with secondary antibodies (1:4000, Cell Signal Technology, Danvers, MA, USA) for 4 hrs at 4 °C. After being washed with TBST, proteins in PVDF membrane bands were detected with luminol reagent (Santa Cruz, USA). GAPDH expression was used as the internal standard. The protein bands were observed and captured with a scanner (HP Deskjet F2288, China). The images were analyzed by Quantity One software.

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Manufacturer protocol

Download the product protocol from Abcam for Recombinant Anti-Vimentin antibody [EPR3776] - Cytoskeleton Marker (ab92547) below.

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