Antibody purification

Buffers and Reagents:

Column: HiTrap NHS-activated HP 1 ml

Coupling buffer - 0.2 M NaHCO₃ , 0.5 M NaCl, pH 8.3

Buffer A - 0.5 M Ethanolamine, 0.5 M NaCl, pH 8.3

Buffer B - 0.1 M Sodiumacetate, 0.5 M NaCl, pH 4.0

1 mM HCl

2 M glycine-HCl pH 2.0

0.1% PBS/NaN₃

PBS

50 mM glycine-HCl pH 2.75

50 mM TAE pH 11.5

Buffer A - 0.5 M Ethanolamine, 0.5 M NaCl, pH 8.3

Buffer B - 0.1 M Sodiumacetate, 0.5 M NaCl, pH 4.0

Protocol:

Preparation of the peptide: Weigh 5 mg of the peptide and dissolve in 1 ml coupling buffer by vortexing.

Determine the protein concentration by measuring absorption at 280 nm using spectrophotometer.

Coupling to the column-

Fill 5 ml syringe with 1 mM HCl. Couple the column by placing the syringe on the yoke of the column.

Activation of the column-

Activate the column and wash away the isopropanol from the column by passing 6 ml of 1 mM HCl through the column. 

Coupling of the peptide-

Connect the column with two syringes, one on the upper side and other on the lower side. Fill the upper syringe with peptide solution and press it slowly on the column. The upper syringe will be empty and lower will be filled up half. Incubate the column for 1 hour at 4 ^oC. Then press the lower syringe slowly in order to move the solution to the upper syringe. Again incubate the set up at 4 ^oC for 1 hour. Move the peptide solution once every hour between two syringes for up to 4 hours. Incubate the whole set up at 4 ^oC overnight. 

Next day-

Remove the lower syringe and let the peptide solution drop out of the column. Preserve the flow through.

Washing of the column-

Wash the column twice using 1 ml coupling buffer. Preserve the flow through and mix it with the previous one. Mix 1 ml of the flow through with 1 ml of 2 M glycine-HCl pH 2.0 and measure the absorption at 280 nm. 

Determination of the coupling efficiency-

Volume of the coupled peptide solution:

A = A_{280, coupling solution} X V_{loaded volume of coupling solution}

Volume of the not coupled peptide:

B = A_{280, post-coupling wash after acidification} X V_{Volume post-column wash} X 2_{(dilution when acidified)}

Coupling yield, %:

(A-B)/A x 100 = Z

Z% of the peptide solution is coupled to the column. 

Denaturation of the column-

Denature the column by passing 2 ml of buffer A, 2 ml of buffer B and again 2 ml of buffer A. Repeat the sequence thrice and incubate the column at 25 ^oC for 30 minutes. Post 30 minutes, pass 2 ml of buffer B, 2 ml of buffer A and 2 ml of buffer B through the column and repeat the sequence thrice followed by incubation at 25 ^oC for 30 minutes.

Equilibration of the column-

Equilibrate the column by washing it with 10 ml of PBS once. 

Storage of the column-

Add 2 ml of 0.1% PBS/NaN₃ to the column and store the column at 4 ^oC.

Antibody purification-

1. Defrost the bleed and take 10 ml out. Freeze back the rest of the bleed at -80 ^oC.

2. Centrifuge the serum at 40,000 rpm, for 1 hour at 4 ^oC using ultracentrifuge.

L1 = clear, thick layer

L2 = pink, thin layer

3. Remove the L2 layer carefully with pasteur pipette and place it on ice.

4. Wash the coupled column with 10 ml of PBS.

5. Absorb the serum in a 5 ml syringe and place it on the column. Arrange the second syringe at the other end of the column.

6. Move the serum within the syringes every 15 minutes for up to 3 hours at 4 ^oC.

7. Collect the sample from the flow through.

8. Wash the column using 10 ml of PBS.

9. Elute the antibody with 10 ml of 50 mM glycine-HCl pH 2.75 and place the eluate on ice.

10. Again wash the column using 10 ml of PBS.

11. Elute the antibody with 10 ml of 50 mM TAE pH 11.5 and place the eluate on ice.

12. Wash the column using 20 ml of PBS and store the column in PBS/NaN₃ at 4 ^oC.

13. Concentrate the eluates to a volume of 4 ml using 30 kDa Amicon’s membranes. Thereafter, dialyse the eluates in PBS for 2 hours and then in PBS/50% Glycerol overnight at 4 ^oC.