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Recombinant Human Tumor Necrosis Factor
Alpha
(
rHuTNF-a)
Certificate of Analysis and Data Sheet
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
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.
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
