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Recombinant Human Tumor Necrosis Factor Alpha

( rHuTNF-a)

Certificate of Analysis and Data Sheet

   Description:

Recombinant Human TNF-a produced in E.Coli is a single, non-glycosylated, polypeptide chain containing 158 amino acids and having a molecular mass of 17483.77 Dalton.

         The rHuTNF-alpha is purified by standard chromatographic techniques.

 Source:

Escherichia Coli.

Physical Appearance:

Sterile Filtered White lyophilized (freeze-dried) powder.

Formulation & packaging:

    The protein was lyophilized after extensive dialysis against 5mM Tris buffer pH = 8.

Solubility:

Lyophilized rHuTNF-a, although stable at room temperature, should be stored desiccated below 0°C. Reconstituted rHuTNF- a  is best stored refrigerated at 4°C

  Purity:

Greater than 99.0% as determined by:

          (a) Analysis by RP-HPLC (See Figure1).

(b) Anion-exchange FPLC.

     (c) Analysis by reducing and non-reducing SDS-PAGE Silver Stained (See Figure 2).

(Limit of acceptance:³98.0%. No more than 2% total impurities; no single impurity greater than 1%)

     Stability:

Lyophilized rHIV-I, although stable at room temperature, should be stored desiccated below 0°C. Reconstituted rHIV-I is best stored refrigerated at 4°C.

  Amino Acid Composition:

        In total agreement with the expected amino acid composition of native human TNF- a.

  Amino acid sequence:

The sequence of the first five N-terminal amino acids was determined and was found to be Met-Val-Arg-Ser-Ser, conforming the sequence of native human TNF- a.

  Dimers and aggregates:

Less than 1% as determined by silver-stained SDS-PAGE gel analysis.

 Biological Activity:

ProSpec’s rHuTNF-alpha is fully biologically active when compared to standard.

The ED50 as determined by the cytolysis of murine L929 cells in the presence of Actinomycin D is < 0.05ng/ml, corresponding to a Specific Activity of 2.2x10⁷ IU/mg.

 Endotoxin:

Less than 0.1 ng/µg (IEU/µg) of rHuTNF- a.

 Protein content:

 Protein quantitation was carried out by two independent methods:

                1.      UV spectroscopy at 280 nm using the absorbency value of 1.234 as the extinction         

coefficient for a 0.1% (1mg/ml) solution. This value is calculated by the PC GENE

          computer analysis program of protein sequences (IntelliGenetics).

 2. Analysis by RP-HPLC, using a calibrated solution of TNF- a  as a Reference Standard.

 Usage:

This material is offered by Gentaur for clinical applications and supplied with a clinical grade certificate of Belgian authorities