FBS Dialysis Protocol: How to Prepare Dialyzed Fetal Bovine Serum

Standard FBS contains glucose, amino acids, nucleotides, vitamins and other small molecules. For SILAC proteomics, ¹³C metabolic flux analysis, metabolomics and selection systems based on nucleotide metabolism, these molecules interfere: they dilute isotope labels or mask the metabolite you want to measure. Dialyzed FBS has these small molecules removed while keeping albumin, growth factors and other proteins.

This protocol describes dialysis of FBS in the lab. For larger volumes and documented lots, SeamlessBio also offers dialysis as a serum processing service. Background on applications: What is dialyzed FBS?

How serum dialysis works

Serum is placed in a semi-permeable membrane (dialysis tubing or cassette) and immersed in a large volume of buffer.

• Molecules smaller than the membrane’s molecular weight cut-off (MWCO) diffuse out into the buffer until the concentrations equalise.
• Larger molecules stay inside: albumin (approx. 66 kDa), transferrin (approx. 80 kDa), IgG (approx. 150 kDa) and most growth factors.

Changing the buffer several times lowers the small molecules to very low residual levels.

Choosing the cut-off: – 10 kDa is the most common choice for dialyzed FBS. It reliably removes amino acids, glucose and nucleotides. – 3.5 kDa retains smaller peptides and small proteins, but takes longer.

Materials

• FBS, e.g. FBS Standard or FBS Premium, thawed at 2–8 °C
• Dialysis tubing or cassettes, MWCO 10 kDa (alternatively 3.5 kDa)
• Dialysis buffer: sterile 0.9 % NaCl (0.15 M) or PBS without Ca²⁺/Mg²⁺, pre-cooled to 2–8 °C
• Clips for dialysis tubing, large beaker or container, magnetic stirrer
• Cold room or refrigerator (2–8 °C)
• Filters: 0.45 µm prefilter and 0.2 µm sterile filter, low protein binding (PES)

Step-by-step protocol

1. Prepare the membrane

1. Cut the dialysis tubing to length. Leave room for clips and for a volume increase of roughly 10–20 %.
2. Prepare the membrane as the manufacturer instructs. Many membranes must be rinsed or soaked in water or buffer to remove glycerol or preservatives.

2. Fill

3. Close one end of the tubing with a clip and fill it with the thawed serum.
4. Remove most of the air, but leave some headspace: water flows into the bag during dialysis because of the high protein concentration in serum.
5. Close the second end.

3. Dialyse

6. Place the filled tubing in at least 50 times its volume of cold buffer (e.g. 100 mL serum in at least 5 L buffer).
7. Stir slowly at 2–8 °C. The tubing should float freely and must not be caught by the stir bar.
8. Change the buffer 3–4 times over 24–48 hours. A common schedule is after 4 h, after 8–12 h (overnight) and after a further 12–24 h.

4. Harvest

9. Take out the tubing, rinse the outside briefly with buffer and open one end.
10. Transfer the serum to a sterile container and note the final volume. Because of the water influx, the serum is now somewhat diluted.

5. Sterile-filter

11. Prefilter through 0.45 µm, then sterile-filter through 0.2 µm. The dialysis step is not sterile, so this filtration is required before cell culture.

6. Aliquot and store

12. Aliquot and store at −20 °C. Label as “dialyzed FBS”, with MWCO, date and lot.

7. Quality control

13. Measure glucose and, for SILAC, the relevant amino acids (e.g. arginine, lysine) in the dialyzed serum. They should be at or near the detection limit.
14. Check osmolality and total protein. They show whether dilution or salt imbalance needs correcting.
15. Test growth of your cell line in the final medium before starting the experiment.

Practical tips

• The buffer defines the salt composition. Dialysis against Ca²⁺/Mg²⁺-free PBS also removes free calcium and magnesium from the serum. In most cases the culture medium supplies these ions.
• Supplement the medium. Dialyzed FBS lacks amino acids, glucose and nucleosides. Your medium must provide them, either defined or labelled for SILAC.
• Expect slower growth. Some cell lines grow more slowly in dialyzed FBS. Increasing the serum concentration slightly or adapting the cells gradually can help.
• Work cold. Dialysis at 2–8 °C reduces protein degradation and microbial growth during the long incubation.

Applications

• SILAC proteomics: removes unlabelled arginine and lysine, so the cells incorporate the heavy isotopes efficiently.
• ¹³C metabolic flux analysis and metabolomics: removes background glucose, glutamine and other metabolites.
• Selection systems: for example HAT and other nucleotide metabolism-based selections, where serum nucleosides interfere.
• Nutrient and drug uptake studies: defined concentrations of small molecules in the medium.

FAQ

What is dialyzed FBS? FBS from which small molecules (amino acids, glucose, nucleotides, vitamins) have been removed by dialysis, while proteins such as albumin and growth factors are retained. See What is dialyzed FBS?

Which molecular weight cut-off should I use for FBS dialysis? 10 kDa is the most common choice. A 3.5 kDa membrane retains more small proteins and peptides but takes longer.

How long does FBS dialysis take? Typically 24–48 hours at 2–8 °C with 3–4 buffer changes.

Does dialysis remove hormones from serum? Partly. Free small hormones can pass the membrane, but many steroid and thyroid hormones are bound to proteins such as albumin and stay in the serum. To remove hormones specifically, use charcoal stripping.

Is dialyzed FBS sterile? Only if it is sterile-filtered after dialysis. The dialysis itself is not a sterile process.

Can I buy dialyzed FBS instead of preparing it? Yes. SeamlessBio dialyses FBS and other sera on request as part of our serum processing service, from 100 mL. Prices and lead times on request – request a quote.

Related: What is Dialyzed FBS? · Serum Processing Services · FBS Charcoal Stripping Protocol · Fetal Bovine Serum · Serum-Free Systems