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Recombinant Human Granulocyte Macrophage Colony Stimulating Factor GM CSF
(mGMP-rHuGM-CSF)

Description:

mGMP Recombinant Human GM-CSF produced in E.Coli is a single, non-glycosylated, polypeptide chain containing 127 amino acids and having a molecular mass of 14477 Dalton. rHuGM-CSF is purified by proprietary chromatographic techniques.

Source:

Escherichia Coli.

Physical Appearance:

Sterile Filtered White lyophilized (freeze-dried) powder.

Formulation & Packaging:

The protein was lyophilized after extensive dialysis against 2mM sodium phosphate buffer pH= 7.4±0.1.

Solubility:

The lyophilized rHuGM-CSF is very soluble in water and most aqueous buffers below and above the isoelectric point (pI=4.95).

Stability:

Lyophilized mGMPrHuGM-CSF although stable at room temperature, should be stored desiccated below 0°C. Reconstituted rHuGM-CSF 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
human GM-CSF.

Amino acid sequence:

The sequence of the first five N-terminal amino acids was determined and was found to be Ala-Pro-Ala-Arg-Ser, conforming the sequence of native human GM-CSF. N-terminal methionine has been completely removed enzymatically.

Dimers and aggregates:

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

Biological Activity:

mGMPrHuGM-CSF is fully biologically active when compared to standard.The ED50 as determined by the dose-dependant stimulation of the proliferation of human TF-1 cells (human erythroleukemic indicator cell line) is 0.1 ng/ml, corresponding to a Specific Activity of 9x106 IU/mg or 2 800 000 IU/ vial of 300 ug

Endotoxin:

Less than 0.1 ng/µg (IEU/µg) of mGMPrHuGM-CSF.

Protein content:

Protein quantitation was carried out by two independent methods:

1. UV spectroscopy at 280 nm using the absorbency value of 0.963 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 standard solution of GM-CSF as a Reference Standard.

Usage:

This material is offered by Gentaur for research, laboratory or further manufacturing purposes adn DC culture

Cell lines:

Hematopoietic cell lines K562, human erythroleukemia cell line (HEL), HL60, TF-1, Jurkat, Daudi, CEM, and ML1 (ATCC, Manassas, VA, USA) were cultured in the presence of 10% fetal bovine serum (FBS) (Euroclone, Milano, Italy) in Iscove's modified Dulbecco's medium (IMDM) or RPMI (Euroclone, Milano Italy) and 5% CO2 in a humidified atmosphere of 5% O2 in air at 37°C. TF-1 required the addition of 10 ng/ml of human granulocyte-macrophage colony-stimulating factor (GM-CSF, Leukomax; Schering-Plough, Basel, Switzerland).

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