Desmin, chicken gizzard, 250 µg
Desmin, chicken gizzard, 250 µg
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Desmin, chicken gizzard, 250 µg
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Key Features
- Purified chicken gizzard desmin
- Protein standard
Product description
| Quantity | 250 µg |
|---|---|
| Storage | Lyophilized at 2-8°C; reconstituted at -20°C (avoid freeze/thaw cycles) |
| Intended use | Research use only |
| Source | Chicken gizzard |
| Molecular weight | 53 kDa |
| Isoeletric point | pI 5.4 |
| Purity | > 98% (determined by SDS gelelectrophoresis) |
| Reconstitution | Reconstitute with 200 µl distilled water (final volume 250 µl). Final solution: 10 mM sodium phosphate buffer pH 7.5, 6 M urea, 2 mM DTT, 1 mM EDTA, 10 mM methylammonium chloride; Protein concentration: 1 mg/ml (determined by Bradford method). |
| Application | Protein standard in 1D and 2D SDS gelelectrophoresis, immunoassays and immunization |
Background
Protein standard for immunoblotting, immunization and immunoassays.
Reconstitution to filaments is performed by dissolving in 6 M urea buffer (see above) at concentrations of approx. 0.5 mg/ml. 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 WW (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 WW (1985). J Cell Biol 101, 1826-1841
- Hatzfeld M et al. (1987). J Mol Biol 197, 237-255
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FAQs
Our chicken Desmin was purified according to the following protocol:
- Storage of chicken gizzard in liquid Nitrogen at -196°C (-320 °F).
- Homogenization in salt solution.
- Solubilization in 6-9 M urea solution.
- Ion exchange chromatography in 6-9 M urea solution for > 12 h.
- Elution with increasing salt concentration.
- HPLC purification in 0.1% trifluoroacetic acid and acetonitril at pH 2.0.
- Vacuum rotation and dialysis.
- Filtration through 0.45 µm filter.
- Lyophilization.
Our chicken Desmin was purified using strong denaturing conditions and HPLC:
Raw material: frozen chicken gizzards obtained from the local abattoir, Mannheim / Germany;
Raw material: frozen chicken gizzards obtained from the local abattoir, Mannheim / Germany;
- Homogenization in buffer I (50 mM Imidazol, pH 7.4; 2 mM DTT, 50 µM Pefabloc).
- Preparation of cytoskeletal material (several wash and centrifugation steps in buffer I).
- Extraction of cytoskeletal material with urea (buffer I plus 8 M urea).
- Chromatographic separation of urea extract on Sepharose S in presence of urea (elution of desmin containing fractions with NaCl-gradient: 100-500 mM NaCl), concentrating and dialysis of desmin-containing fractions.
- Re-chromatography of desmin-containing fractions on DEAE cellulose, elution with 50 mM NaCl.
- Concentrating desmin-containing fractions by precipitation with 100% ethanol.
- Chromatography by HPLC (reverse phase: TFA/acetonitril).
- Evaporation of dissolving solution.
- Analysis by SDS-PAGE and Western blotting.
- Determination of protein concentration by Bradford method.
- Solubilization in urea buffer and lyophilization.