Vimentin, human recombinant
Referentie 62015
Formaat : 250µg
Merk : Progen
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Telefoonnummer : +1 850 650 7790
| Quantity | 250 µg |
|---|---|
| Storage | Lyophilized at 2-8°C; reconstituted at -20°C (avoid freeze/thaw cycles) |
| Intended use | Research use only |
| Source | Human recombinant, produced in E. coli |
| Molecular weight | 57 kDa |
| Isoeletric point | pI 5.3 |
| Purity | > 95% (determined by SDS gelelectrophoresis) |
| Reconstitution | Reconstitute with 175 µl distilled water (final volume 250 µl). Final solution: 30 mM Tris/HCI pH 8, 9.5 M urea, 2 mM DTT, 2 mM EDTA, 10 mM methylammonium chloride; Protein concentration: 1 mg/ml |
| Application | Protein standard in 1D and 2D SDS gelelectrophoresis, immunoassays and immunization |
Protein standard for immunoblotting, immunization and immunoassays.
Reconstitution to filaments: after vimentin is dissolved in 9.5 M urea buffer (see above), protofilaments and filament complexes are obtained by dialyzing the resulting polypeptide solution stepwise to a concentration of 4 M urea and then to low salt condition (50 mM NaCl, 2 mM dithiothreitol, 10 mM Tris-HCl, pH 7.4).
For immunization purposes, the solution can be further dialyzed against PBS (phosphate buffered saline, e.g. Dulbecco's PBS).
- Hatzfeld M. and Franke W.W. (1985). J. Cell Biol. 101, 1826-1841
- Hatzfeld M. et al. (1987). J. Mol. Biol. 197, 237-255
For immunization purposes, the solution can be further dialyzed against PBS (phosphate buffered saline, e.g. Dulbecco's PBS).
- Hatzfeld M. and Franke W.W. (1985). J. Cell Biol. 101, 1826-1841
- Hatzfeld M. et al. (1987). J. Mol. Biol. 197, 237-255
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Our human recombinant vimentin was lyophilized in urea buffer. Vimentin other intermediate filament proteins are insoluble under physiological buffer conditions (like PBS) or low salt conditions. This is a prominent feature of this protein family. There is some solubility in Tris-buffer, however, at very low protein concentration.
To obtain filaments from Cat. No. 62015 we suggest the following procedure:
- Reconsitute the lyophilized material in distilled water to a protein concentration of 1 mg/ml and a urea concentration of 9.5 M.
- Dialyze against 6 M urea buffer (down to 6 M urea vimentin is still present in the monomeric form) for at least 2 h at RT.
- Dialyze against 4M urea buffer (at 4 M urea dimers and protofilaments are formed) for at least 4 h at RT or overnight at 4°C.
- Dialyze to low urea/low salt buffer (at least 2 buffer changes to obtain intermediate filaments) containing 50 mM NaCl, 2 mM dithiothreitol, 10 mM Tris-HCl, pH 7.4, or filament forming buffer (suggested e.g. by Herrmann et al. 1996, J Mol Biol 264, 933-953) containing 25 mM Tris-HCl pH 7.5, 0.1 mM dithiothreitol, 160 mM NaCl- for immunization purposes dialyze to e.g. PBS.
- Dialysis in steps against large buffer volumes is important in order to obtain filament complexes and avoid formation of amorphous aggregates.
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