Reconstituting and Aliquoting Mouse GDF-5 (R&D Systems)

Youssef Farhat, Written 10/30/13, Last updated 10/30/13

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Introduction

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.

Materials

• 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)

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Procedure

1. Prepare and Aliquot Reconstitution Solution

1. Under the hood, combine the following into a 50 mL centrifuge tube to make Reconstitution Solution:
   1. 11.8 mL of UltraPure water
   2. 160.0 µL of BSA stock (7.5%, or 0.075 g/mL)
   3. 40.0 µL of HCl stock (1.2 M)
2. Sterile-filter the Reconstitution Solution
3. Aliquot the Reconstitution Solution into 1.5 mL Eppendorf tubes, 0.6 mL per tube
4. 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

1. 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).
2. Briefly vortex the GDF-5 solution to ensure complete dissolution.
3. 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]).
4. Label the tubes, lightly centrifuge the droplets of reagents down to the bottom of the tubes, and store at -80°C.

Citing this Protocol

Farhat, Y. “Reconstituting and Aliquoting Mouse GDF-5.” The Protocol Place. Oct 2013. Accessed on mm/dd/yyyy. <http://protocol-place.com/>