Youssef Farhat, Written 6/5/13, Last updated 10/30/13
The purpose of this protocol is to reconstitute and aliquot a vial of human, recombinant GDF-5. GDF-5 is a growth factor that plays an important role in development, wound healing, and a number of disease processes. Hence, there is a need to study the effect of this growth factor on a number of cells under various conditions.
Lyophilized (freeze-dried), purified GDF-5 can be obtained from a number of commercial sources. In this protocol, we describe the process of reconstituting and aliquoting GDF-5 obtained from BioVision to a final concentration of 500 ng/µL.
The GDF-5 used here is reconstituted in solution containing 20 mM HOAc (Acetic Acid, Molecular Weight 60.06 g/mol, Density: 1.05 g/mL), as recommended by the manufacturer. Other growth factors and reagents can be reconstituted in a similar manner; however, it is important to follow the manufacturer’s recommendations regarding the contents of the Reconstitution Solution used and the appropriate storage of all reagents.
- 50 µg of Recombinant Human GDF-5 (BioVision, 4667-50)
- UltraPure™ DNase/RNase-Free Distilled Water (Gibco, #10977-015)
- Acetic Acid (J.T. Baker, 95-2233)
- Sterile filter mechanism of choice (i.e. syringe filter, vacuum filter)
1. Prepare and Aliquot Reconstitution Solution
- Under a fume hood, combine the following into a 50 mL centrifuge tube to make the Reconstitution Solution (20 mM HOAc):
- 11.44 µL of Acetic Acid
- 10 mL of UltraPure water
- Sterile-filter the Reconstitution Solution in a culture hood
- Aliquot the Reconstitution Solution into 1.5 mL Eppendorf tubes, 1 mL per tube
- Label and store all but one of the tubes at -20°C for reconstitution in the future.
2. Reconstitute GDF-5 and Aliquot
- Under the culture hood, add 0.5 mL of sterile Reconstitution Solution to the vial containing 50 µg of GDF-5. Dissolve the GDF-5 by pipetting up and down several times, or by vortexing briefly and then spinning down the solution on a benchtop centrifuge. Final concentration: 100 ng/µL.
- Working under the hood again, aliquot the GDF-5 as desired into 1.5 mL Eppendorf tubes (for my experiments, I prepared 50 and 100 µL aliquots [containing 5 and 10 µg GDF-5, respectively]).
- Label the tubes, lightly centrifuge the droplets of reagents down to the bottom of the tubes, and store the GDF-5 at -20°C.
Citing this Protocol