Analysis and purification of IgA from Shanghai Xinfan Technology Lecture - Huaqiang

(1) Preparation of materials and reagents

1. DEAE52

Cellulose

2.0.10Mol/L ZnSO

₄

Liquid ZnSO

₄

·7H

₂

O 2.88g Add water to 1 000.00ml
3.

Saturated ammonium sulfate solution

4.10% EDTA—

Na

5. SephadexG200
6.0.01Mol/L pH7.4 PBS solution
7.0.10 Mol/L pH6.4 PBS solution
8

. serum

(two) method of operation

1. Take serum plus an equal amount of 0.1Mol/L ZnSO

₄

The solution was adjusted to pH 7.0, stirred at room temperature for 1 h, and centrifuged to precipitate.
2.

In the supernatant

Add an equal amount of saturated ammonium sulfate solution, mix and let stand for 30 min or overnight in the refrigerator.
Centrifuge at 3.10 000 r/min for 10 min and remove the supernatant.
4. The precipitate was dissolved in 4 ml of 10% EDTA-Na solution.
5.

Dialysis demineralization in normal saline

.
6. Through DE

₅₂

Column with 0.01Mol/L pH7.4P

B solution eluted protein (IgG) discarded

.
7. Elected with 0.1Mol/L pH6.4PB solution

IgA

.
8. SephadexG200

Column, lotion is 0

.01Mol/L pH7.4PBS (0.14Mol/L NaCl), collect the eluent to measure OD

₂₈₀

Value, taking the first peak is the purified IgA.
9. Dialysis, concentration

Identification