Biotechnology Reagents (IGFs) >> Product Support >> Application Notes >> #1250: Handling of Novozymes GroPep LONG®EGF and LONG®TGF-α

Reconstitution of the peptide:
LONG®EGF and LONG®TGF-α is supplied lyophilized in a glass vial in an atmosphere of nitrogen at a slight vacuum (-25kPa). Care should be taken when opening the vial to equilibrate the contents with ambient pressure to avoid losses. An air-filled syringe may be introduced through the bung to equalize the pressure before opening.

Step 1. Dissolve vial to a concentration of 1 mg/ml in 10mM HCl to give a stock solution that may be stored at -20°C or -70°C. If long-term storage is required, store lyophilized at -70°C.

IMPORTANT: Irrespective of size, always reconstitute the entire vial in situ and then aliquot. Each vial contains the net amount of peptide indicated on the label but ~15 - 20 % more by mass due to the residual moisture retained through the freeze drying process. DO NOT estimate peptide content by weight or gravimetric means as this will underestimate the actual peptide content by ~15 - 20 %.

Step 2. To use, dilute further to 0.1 mg/ml in 10mM HCl (working solution). At this concentration BSA or carrier protein may be present but phosphate buffered saline (PBS) or buffer containing phosphate should not be present as this causes the LONG®EGF or LONG®TGF-α to precipitate out of solution.

Step 3. This working solution can now be diluted into PBS or other buffered solution or media but preferably with a carrier protein (eg BSA 1- 5 mg/ml, or FBS) or other substance to prevent adsorption losses.

LONG is a trade mark owned by Novozymes GroPep Limited

Filter Sterilization of LONG®EGF or LONG®TGF-α:

If sterilization is required, the peptide solution should be filter sterilized using a membrane with low protein binding; an example of this is the Millipore Millex-GV Cat. No. SLGV025LS.

Note that extreme care must be exercised in filtering dilute protein solutions because of the likelihood of adsorption to the filter. Growth factors require the same care in handling. Filtration for sterility is best done on either the 1 mg/ml stock solution or the 0.1 mg/ml working solution diluted in 10 mM HCl. It may be possible to filter more dilute solutions if a carrier protein is present but caution should be exercised.

Important Recommendations:

*Do not add the peptide to low protein or protein free media prior to filter sterilization.

*Use a filter membrane with low protein binding characteristics.

*Filter sterilize the LONG®EGF or LONG®TGF-α separately at a concentration of 0.1 mg/ml (diluted in 10 mM HCl) or greater.

* NOT FOR USE IN HUMANS


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