Recombinant murine Tumor Necrosis Factor-alpha

( rmTNF-a )

Certificate of Analysis and Data Sheet

 

       ×         Description:

Recombinant murine TNF-a produced in S.Lividans is a single, glycosylated, polypeptide chain containing 156 amino acids and having a molecular mass of 17301.32 Dalton.

         The rmTNF-alpha is purified by standard chromatographic techniques.

 

       ×         Source:

Streptomyces Lividans

 

       ×         Physical Appearance:

Sterile Filtered White lyophilized (freeze-dried) powder.

 

       ×         Formulation & packaging:

The protein was lyophilized from a concentrated (1mg/ml) sterile solution containing 20mM Tris-HCL, pH=8, 5mM MgCl2 and 10% glycerol.

     

       ×         Solubility:

The lyophilized murine TNF- a is very soluble in water and most aqueous buffers below and above the isoelectric point.

 

       ×         Stability:

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

 

       ×         Purity:

Greater than 99.0% as determined by:

(a) Analysis by RP-HPLC.

(b) Anion-exchange FPLC.

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

 

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

 

       ×         Amino Acid Composition:

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

 

       ×         Amino acid sequence:

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

 

       ×         Dimers and aggregates:

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

 

       ×         Biological Activity:

Our rmTNF-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  9x10⁸ IU/mg.

 

       ×         Endotoxin:

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

 

       ×         Protein content:

 Protein quantitation was carried out by two independent methods:

 

1. UV spectroscopy at 280 nm.

 

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

 

      ×        Usage:

This material is offered for research, laboratory or further manufacturing purposes.