Ammonium Sulphate Fractionation of Antibodies
- Protocols
Dr AF Saeed- 0
- 6 minutes read
Ammonium Sulphate Fractionation of Antibodies (Crude Protein Purification)
Abstract
A “cut” with ammonium sulphate was the go-to procedure for isolating IgG and other blood proteins until Protein A (rabbit) and Protein G (rodent) were widely used for the purpose. Ammonium sulphate’s presence decreases proteins’ effective solubility by competing for binding sites on the protein’s surface. Centrifugation may then be used to separate the precipitated proteins. Most species’ IgG may be precipitated using a 50% ammonium sulphate solution, while concentrations as low as 40% have been employed successfully. Using ammonium sulphate alone does not result in a pure antibody fraction; rather, it is the first stage of a multistep antibody purification technique due to the possibility of other proteins being “trapped” inside the aggregated protein.Reagents
Saturated ammonium sulphate (more than 99 per cent purity)Sodium phosphate-buffered saline
Supernatant from cell cultures, serum, or ascites should be handled carefully.10 mM Tris (pH 6.5) buffer
Equipment
Refrigerated (or cold room-housed) centrifuge with enough capacity to handle the amount at handDialysis tubing (MWCO of 10 or 25 kDa).
Filters (0.2 m)Put the filter in a bottle, Nalgene, or syringe. The decision will be influenced by the quantity of data being processed.
pH measuring deviceUV spectrophotometer with a stirring plate
METHOD
1. Centrifuging at 3000g for 30 minutes at 4°C will remove insoluble fragments from serum, ascites, or cell culture supernatant.2. Place the supernatant in a beaker and add 25% (w/v) saturated ammonium sulphate slowly.
3. Keep in an incubator at 4 degrees Celsius for 5 to 15 hours.4. Spin at 3000 g for 30 minutes at 4 degrees Celsius.
5. Decant the supernatant into a clean beaker, taking care not to spill it.
6 Mix in enough saturated ammonium sulphate
(about a third of the volume of the supernatant) to lower the final concentration to 50%.7. Let it sit at 4 degrees Celsius for 5–15 hours.8. Then, centrifuge at 3000g for 30 minutes at 4°C.
9. The supernatant should be discarded. The pellet should be carefully resuspended in a volume that is 30%-50% of its original size. Fill up a length of dialysis tubing (10-25 kDa MWCO) with the whole sample. Use dialysis tubing with double the volume of the sample to account for the expansion of the fluid during dialysis and to prevent excessive foaming.
10. Dialyze for 24 to 48 hours against at least three changes of 1x PBS. Centrifugation may get rid of any flocculent matter. However, if further antibody purification is to be accomplished through ion-exchange chromatography (such as DEAE), a dialysate with a lower ionic strength, such as 10 mM Tris buffer (pH 6.5) or a comparable buffer, should be employed.
11. After dialysis is finished, the protein concentration may be calculated by measuring the optical density at 280 nm. A280 = 1.35 for IgG = 1 mg/mL.
Protein A/G purification, anion-exchange chromatography using diethyl aminoethyl (DEAE)-Sepharose, and other techniques (affinity chromatography, size exclusion, and other chromatographic methods) may all be used to further purify this crude antibody fraction. The IgG fraction should be filtered using a 0.45-m filter and stored at 4°C if it is to be kept for an extended period of time without additional workup.
