Isolation and purification of IgG
Isolation and purification of IgG
IgG isolation and purification experiments can be used to: prepare enzyme labeled antibodies or fluorescent antibodies.
Operation method
Ammonium sulfate precipitation
Principle
Ammonium sulfate precipitation can be used to concentrate and partially purify proteins from large quantities of crude preparations. The major immunoglobules can be separated from the sample by this method, and it is a common method for immunoglobulin separation. High concentrations of salt ions in protein solutions can compete with proteins for water molecules, thereby disrupting the hydration membrane on the protein surface and reducing its solubility, causing it to precipitate out of solution. The solubility of various proteins is different, and thus different concentrations of salt solutions can be utilized to precipitate different proteins. This method is called salting out. Salt concentration is usually expressed in terms of saturation. Ammonium sulfate is the most widely used because of its high solubility, small temperature coefficient, and the fact that it does not easily denature proteins.
Materials and Instruments
Animal serum samples Move I. Material and reagent preparation Caveat Generally concentrated to a concentration of 1% or more, and then divided into vials for lyophilization and preservation, or add 0.01% thimerosal in the ordinary refrigerator or low-temperature refrigerator preservation, pay attention to prevent repeated freezing and thawing. Common Problems To purify IgG in small quantities of a few tens of ml, protein A or protein G can be used, and hydrophobic columns can also be used for purification; in large quantities of hundreds of ml, you can use streamline protein A. Estimate the volume of the column you need according to the amount of loading beforehand. For more product details, please visit Aladdin Scientific website.
Ammonium sulfate NH4OH PB solution BaCl2 solution Nano solution Elution solution Physiological saline
Dialysis bag Centrifuge Beaker Stirring rod
1. Animal serum
2. Saturated solutions of ammonium sulphate
Ammonium sulfate: 800 g~850 g
H2O: 1 000 ml
Heat until the majority of the solute is dissolved, filter while hot, leave at room temperature overnight, then adjust pH to 7.0 with 28% NH4OH (no pH adjustment is possible).
Note: Ammonium sulfate is preferable to the best quality, because the inferior product contains a small amount of heavy metals on the protein sulfhydryl group has an effect. If the heavy metals must be removed from the inferior product, H2S can be passed into the solution, which can be left overnight and filtered, and the H2S can be evaporated by heating.
3. 0.01 mol/L pH 7.4 PB solution
Liquid A: 0.10 mol/L NaH2PO4 liquid
NaH2PO4-2H2O: 15.60 g
Add H2O to 1 000 ml
Solution B: 0.10 Mol/L Na2HPO4 solution
Na2HPO4-12H2O: 35.80 g
Add H2O to 1 000 ml
Just take 19 ml of liquid A and 81 ml of liquid B and add water to 1,000 ml.
4. 1% BaCl2 solution
5. Nasher's solution
HgI: 115.00 g
KI: 80.00g
Add H2O to 500.00 ml
Dissolve and filter, then add 20% NaOH 500.00 ml and mix.
6. 0.50 mol/L HCl solution and 0.50 mol/L NaOH solution
7. Elution solution
0.03 mol/L NaCl solution.
8. Dialysis bag (or cellophane)
II. Methods of operation
1. Take 20 ml of serum, add 20 ml of saline, then add 10 ml of ( NH4 ) 2SO4 saturated solution drop by drop to make 20% ( NH4 ) 2SO4 solution, stirring while adding, mixing thoroughly, and then leave for 30 min.
2. Centrifuge at 3 000 r/min for 20 min and discard the precipitate to remove fibrin.
3. Add 30 ml of ( NH4 ) 2SO4 saturated solution to the supernatant to make a 50% ( NH4 ) 2SO4 solution, mix thoroughly and leave for 30 min.
4. Centrifuge at 3 000 r/min for 20 min and discard the supernatant.
5. Add 20 ml of saline to the precipitate to dissolve it, then add 10 ml of ( NH4 ) 2SO4 saturated solution to make 33% ( NH4 ) 2SO4 solution, mix thoroughly and leave for 30 min.
6. Centrifuge at 3 000 r/min for 20 min and discard the supernatant to remove albumin. Repeat step 5 2 to 3 times.
7. Dissolve the precipitate in 10 ml of saline and place in a dialysis bag.
8. Desalination by dialysis, overnight in normal water, then in saline at 4°C for 24 h, with several fluid changes.
Check SO42- in the dialysate with 1% BaCl2 or NH4 with Nasher's reagent (take 3~4 ml of dialysate, add 1~2 drops of reagent, and the presence of brick-red color is considered to be the presence of NH4 ) until there is no SO42- or NH4 present. SephadexG25 or electrodialysis can also be used to remove salt.9. Centrifugation to precipitate (to remove heterogeneous proteins), the supernatant that is the crude extract of IgG (that is, gamma globulin, such as 36% saturated ammonium sulfate precipitation serum product that is euglobulin, Euglobin, containing gamma globulin).
10. Pass through a DEAE-cellulose chromatography column. Elute with 0.01 mol/L pH 7.4 PBS (0.03 mol/L NaCl) and collect the eluate. Sephadex G150 or G200 columns can also be used.
11. Proteins and their quantitative identification.
12. The purity of IgG can be determined by one of the following methods.
(1) Zone electrophoresis
Either slide agar or acetate membrane electrophoresis is possible. After electrophoresis of the added samples, only one band appears at the migration site of γ-globulin. When operating, at the same time, whole serum samples can be used, different concentrations of (NH4)2SO4 saline samples for electrophoresis, for comparison.
(2) Agar biphasic double diffusion identification
Pre-prepare the anti-IgG serum obtained from immunization of heterozygous animals with this IgG. Biphasic double diffusion of the IgG with the anti-IgG serum results in a precipitation line between the two sample wells if the IgG is purified.
(3) Immunoelectrophoretic identification
The wells are filled with the samples to be tested, and after electrophoresis, anti-IgG serum is added to the tank and the agar is diffused for 24h, and the results are observed. If the extracted IgG is pure, only one curved precipitation line appears and the precipitation line is located in the γ-globulin region. Immunoelectrophoresis of both whole serum and antiserum antibodies must be performed for this identification for comparison.
(4) Disc electrophoresis identification
Disc electrophoresis was performed simultaneously with whole serum samples and purified samples. Whole serum samples showed tens of bands on disk electrophoresis, while purified IgG showed only one band.
13. Concentration and preservation of IgG
(1) Concentration of IgG
(2) Preservation of IgG
Generally concentrated to a concentration of 1% or more, and then dispensed into vials for lyophilization and preservation, or add 0.01% thimerosal in the ordinary refrigerator or low-temperature refrigerator preservation, taking care to prevent repeated freezing and thawing.