Table 2.
Instructions for the preparation of fetuin aliquots
| Materials | Catalog no. | Supplier | Final concentration |
|---|---|---|---|
| Sterile MilliQ H2O | Base | ||
| Fetal bovine fetuin | F-3385 | Sigma | 10 mg/mL |
| BCA protein assay reagent kit | 23227 | FisherScientific | |
| Spectra/Pro regenerated cellulose Dialysis membranes | MWCO 8000 1231166 | Spectra/Pro Repligen | |
| Procedure: | |||
| • Dissolve fetuin to a 15–20 mg/mL concentration in MilliQ H2O | |||
| • Dialyze the fetuin solution at 4 °C overnight in MilliQ H2O in a 1:50 to 1:100 ratio. Stir gently during all the process. Next day, replace with fresh MilliQ H2O, and dialyze 1–2 more times. Dialysis is important to increase the purity of the fetuin concentrate. | |||
| • Measure the fetuin concentration using a BCA protein Assay Reagent Kit or Nanodrop | |||
| • Dilute fetuin solution to 10 mg/mL with MilliQ H2O | |||
| • Aliquot fetuin into 1.5 mL microcentrifuge tubes (1 mL/tube) | |||
| • Close all the tubes, and puncture a hole in the lid of each tube | |||
| • Freeze until completely solid (several days at −20 °C, overnight at −80 °C or 10 min in liquid nitrogen) | |||
| • Lyophilize the fetuin to dryness at −80 °C. It will take approximately 1 day | |||
| • Seal the holes of the lids with stickers to keep the fetuin dry | |||
| • Fetuin can be stored for long term at −80 °C or at −20 °C for short term use | |||
| We usually prepare 25 g of fetuin to have stock for 3–4 years, but this will depend on how often follicle culture will be performed in your laboratory | |||
| Lyophilization at – 80 °C is a complex process and you need a special equipment | |||