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- Vimentin protein: recombinant syrian hamster
Vimentin protein: recombinant syrian hamster
- Investigating vimentin filament dynamics.
- Identification of vimentin associated proteins.
- Vimentin is an excellent substrate for protein kinases.
Recombinant Syrian hamster vimentin has been expressed in and purified from a bacterial expression system.
Purity is determined by scanning densitometry of the protein on an SDS-PAGE gel. Vimentin is >90% pure.
The protein is supplied in a non-polymerized form. Polymerization to form vimentin intermediate filaments (IFs) can proceed by the addition of NaCl to 150 mM final concentration. V01 has a critical concentration 0.017 mg/ml.
For product Datasheets and MSDSs please click on the PDF links below. For additional information, click on the FAQs tab above or contact our Technical Support department at email@example.com
Perlson, E., Hanz, S., Ben-Yaakov, K., Segal-Ruder, Y., Seger, R. and Fainzilber, M. (2005). Vimentin-dependent spatial translocation of an activated MAP kinase in injured nerve. Neuron 45, 715-726.
Styers, M. L., Salazar, G., Love, R., Peden, A. A., Kowalczyk, A. P. and Faundez, V. (2004). The endo-lysosomal sorting machinery interacts with the intermediate filament cytoskeleton. Mol. Biol. Cell 15, 5369-5382.
Question 1: How do you prevent intermediate filament polymerization as a negative control?
Answer 1: Omission of the NaCl from the vimentin polymerization buffer will prevent polymerization of intermediate filaments (Cat. # V01). The resulting buffer is 5 mM PIPES, pH 7.0.
Question 2: How do I polymerize vimentin for electron microscopy analyses?
Answer 2: We recommend the following polymerization conditions:
- Dilute one vial of vimentin (Cat. # V01; 50 mg total protein at 5 mg/ml) to 0.5 mg/ml by adding 90 ml of Vimentin Subunit Buffer at 4°C.
- Incubate at room temperature for 1h to dissociate aggregates that form during storage.
- Add 3 ml of Vimentin Polymerization Buffer (final NaCl concentration is 150 mM) and mix thoroughly.
- Incubate at 35°C for 30 minutes. This procedure should result in >90% of the vimentin protein polymerizing to produce vimentin intermediate filaments (IFs). Filaments can be used directly in an assay or they can be pelleted by centrifugation at 100,000 x g for 30 minutes at 25°C. Under the above conditions, vimentin filaments are stable at 4°C for at least 1 week.
Then, the IFs can be stained for transmission electron microscopy.
- Dilute 0.5 g/ml vimentin filaments to 0.1 mg/ml concentration in 5 mM PIPES-NaOH pH 7.0, 170 mM NaCl and 0.5% glutaraldehyde.
- Immediately place 5 ml on a formavar coated EM grid (Pella Inc. CA). Allow 30 sec incubation at 24ºC.
- Wick off with 1 cm2 pieces of filter paper.
- Wash twice with Milli-Q water (5 ml each) at 24ºC.
- Add 5 ml of 3% uranyl acetate, leave 30 sec and wick off at 24ºC. If staining is dark, wash at this stage once or twice with Milli-Q water.
- Leave to dry and visualize at about 10,000-12,000X magnification.
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