Youssef Farhat, Written 10/30/13, Last updated 10/30/13
The purpose of this protocol is to reconstitute and aliquot a vial of mouse, 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 R&D Systems to a final concentration of 500 ng/µL.
The GDF-5 used here is reconstituted in solution containing 4 mM HCl and 1 mg/mL of BSA, 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 Mouse GDF-5 (R&D Systems, #853-G5-050)
- UltraPure™ DNase/RNase-Free Distilled Water (Gibco, #10977-015)
- Bovine Albumin Fraction V Solution (7.5%, or 0.075 g/mL) (Gibco, #15260-037)
- 1.2 M HCl Stock Solution (prepared by diluting 10 mL of 12 M HCl [Sigma-Aldrich, # H1758] with 90 mL of deionized water)
- Sterile filter mechanism of choice (i.e. syringe filter, vacuum filter)
1. Prepare and Aliquot Reconstitution Solution
- Under the hood, combine the following into a 50 mL centrifuge tube to make Reconstitution Solution:
- 11.8 mL of UltraPure water
- 160.0 µL of BSA stock (7.5%, or 0.075 g/mL)
- 40.0 µL of HCl stock (1.2 M)
- Sterile-filter the Reconstitution Solution
- Aliquot the Reconstitution Solution into 1.5 mL Eppendorf tubes, 0.6 mL per tube
- Label and store the tubes at -20°C for reconstitution in the future. Save one tube for immediate use and thaw the other tubes as needed for future reconstitutions.
2. Reconstitute GDF-5 and Aliquot
- Under the hood, add 333 µL of sterile Reconstitution Solution to the vial containing 50 µg of GDF-5 and dissolve by pipetting up and down several times (final concentration 150 ng/µL).
- Briefly vortex the GDF-5 solution to ensure complete dissolution.
- Working under the hood again, aliquot the GDF-5 as desired into 1.5 mL Eppendorf tubes (for my experiments, I prepared 25 µL aliquots [each containing 3750 ng GDF-5]).
- Label the tubes, lightly centrifuge the droplets of reagents down to the bottom of the tubes, and store at -80°C.
Citing this Protocol