Western blotting Vimentin

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 5 matching solutions for this experiment

Vimentin Antibody (E-5): sc-373717

Santa Cruz Biotechnology

Upstream tips
Total protein was extracted using radioimmunoprecipitation assay lysis buffer (Beyotime Institute of Biotechnology, Shanghai, China) according to the manufacture's protocol. Briefly, the cells were incubated with the lysis buffer at room temperature for 5 min. Then cell lysates were centrifuged at 13,000 × g for 5 min at room temperature and supernatants were harvested.
Protocol tips
Equal amounts of total protein (20 µg) were separated using 12% SDS-PAGE and subsequently transferred onto nitrocellulose membranes (EMD Millipore, Billerica, MA, USA). Following blocking with 3% bovine serum albumin (Sigma-Aldrich; Merck KGaA) at 4°C overnight, membranes were incubated with keratin rabbit polyclonal antibody (1:400; cat. no. 41723; Signalway Antibody Inc., College Park, MD, USA), GSTA1 monoclonal antibody (1:500; cat. no. sc-100546), E-cadherin rabbit polyclonal antibody (1:500; cat. no. sc-7870), vimentin monoclonal antibody (1:800; cat. no. sc-373717), N-cadherin mouse monoclonal antibody (1:500; cat. no. sc-393933) and β-actin monoclonal antibody (1:1,000; cat. no. sc-130301; all Santa Cruz Biotechnology, Inc., Dallas, TX, USA) at 4°C overnight. Membranes were subsequently incubated with horseradish peroxidase-labeled goat anti-mouse (1:2,000; cat. no. sc-2005) or goat anti-rabbit antibodies (1:2,000; cat. no. sc-2004; both Santa Cruz Biotechnology, Inc.) at 37°C for 1 h.
Downstream tips
Immunoreactive bands were detected using an enhanced chemiluminescence detection kit (Pierce; Thermo Fisher Scientific, Inc.). ImageJ version 1.37 software (National Institutes of Health, Bethesda, MD, USA) was used for quantification, and the experiments were independently performed three times.
Upstream tips
For protein extraction, HK-2 cells were lysed on ice in lysis buffer (Tris 10mM à pH 7.5, NaCl 150mM, EDTA 1mM, EGTA 1mM, SDS 0.1%, NP40 1% and deoxycholate 1%) supplemented with a protease inhibitor cocktail (Complete, Mini, Roche).
Protocol tips
Proteins were separated in a 4–15% SDS-PAGE and blotted on a nitrocellulose membrane (Amersham). Membranes were blocked with TBS-Tween 0.1% supplemented with milk 5% for 30 min. Then, membranes were incubated with primary antibodies (Anti E-Cadherin [1:200, Sc-7870 Santa Cruz], anti Fibronectin [1:500, F3648 Sigma], anti αSMA [1:500, Ab5964 AbCam], anti Vimentin [1:1000, MS-129-P Neomarkers], anti β-Actin (1:5000, A5441 Sigma)) overnight at 4°C followed by incubation with the peroxidase-conjugated secondary antibody (anti rabbit or anti mouse [1:10000, Bethyl]) for 1h.
Downstream tips
Specific bands were detected using SuperSignal West Pico Chemiluminescent Substrate (Thermo Scientific) and the ChemiDoc XRS+ System (Bio-Rad). Protein bands were quantified by densitometry using Image Lab software (version 4.0.1, Bio-Rad) and results are expressed as ratio between target protein and β-Actin.
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.
Protocol tips
-Mouse (1:1500)
--Use clean forceps to gently handle the blot from the corner without creasing the membrane. Do not write on the blot with pen or marker, as the ink
can fluoresce and cause background.
Vimentin Monoclonal Antibody (V9)

Thermo Fisher Scientific

Protocol tips
-Mouse (1:2000)
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