Affinity purification of antibody
Step 1. Chemically cross-link antigen to beads for affinity purification
We use HiTrap NHS-activated HP column for affinity antibody purification. This is a
pre-packed column of N-hydroxy-succinimide (NHS) cross linked to HP Sepharose
beads. NHS reacts with ligands containing amino groups to give a very stable amide
linkage. This column can be connected to FPLC. It comes in 100% isopropanol and once
it is exposed to water-based solution, NHS quickly loses its activity so that antigen has to
be loaded immediately after the column is exposed to water.
Materials:
 HiTrap NHS-activated HP column (1ml) (Amersham, #17-0716-01)
 Antigen (0.5 ~ 10mg in 1 ml; dialyzed against 0.2 M NaHCO3 pH 8.3, 0.5 M
NaCl)
 1mM HCl solution
 Buffer 1: 0.5M ethanolamine pH 8.3, 0.5M NaCl
 Buffer 2: 0.1M acetate pH 4, 0.5M NaCl
Method:
1. Wash HiTrap NHS-HP column with 6 column volume (= 6 ml) 1mM HCl with
flow rate 1 ml/min or 1 drop/3 sec. Keep this flow rate unless specified.
2. Immediately load antigen solution. It has to be immediate otherwise cross-linking
efficiency will dramatically drop.
3. Seal the column and let it stand @RT for 15~30min to cross-link
4. Deactivate remaining NHS. Inject 6 column volumes of Buffer 1, followed by 6
column volumes of Buffer 2. Repeat the washing with Buffer 1 and allow the
column to stand for 15-30 min. Save the first 5 ml to determine cross-linking
efficiency.
5. Wash column with 6 column volumes of Buffer 2, 6 column volumes of Buffer 1,
6 column volumes of Buffer 2, then 6 column volumes of PBS (+ azide).
6. Store at 4 ºC until use.
7. Determine how much antigen cross-linked to the column.
Step 2. Affinity purify specific antibody
Estimation of antibody amount to load onto column:
Maximum 10 % of IgG (or IgY) is expected to be specific antibody (see Antibodies a
laboratory manual, p.291). Equimolar amount to antigen multiplied by 10 will be the
amount of antibody to load. MW of IgG (and IgY) is ~180 kDa.
<example>
To purify Aip1 antibody with NHS-HP column in which 4mg Aip1 is cross-linked:
Aip1 MW 65 kDa, IgY MW ~180 kDa.
4 mg x (180/65) x 10 = 120 mg
Minimum 120 mg IgY fraction should be loaded.
Typically IgY fraction sent from Aves Lab is ~28 mg/ml, >90 % pure IgY.
120 / 28 = 4.3 ml
Minimum 4.3 ml IgY fraction should be used.
Method:
1. Wash column with 6 column volumes of antibody elution solution to omit any
unbound or broke down antigen from column.
2. Inject 1ml 1 mg/ml BSA/PBS and let it stand for 30 min at RT for blocking
column.
3. Dilute IgY fraction to 10 fold in PBS.
4. Load antibody solution to column. Use peristaltic pump to circulate antibody for 1
hr.
5. Wash the column with 10 column volume of 1 mg/ml BSA/PBS. Save unbound
antibody.
6. Elute with 5 column volumes of either 100mM glycine pH2.5 *. Collect 1 ml
elutant each in 1.5 ml tubes and immediately adjust pH with 100µl 1M Tris.
7. Dialyze elutant O/N against PBS.
8. Wash the column immediately with more than 10 column volumes of PBS until
pH is restored. Replace buffer with PBS +azide and store at 4ºC.
9. Add sodium azide to purified antibody and store at 4ºC. Check titer of the purified
antibody and unbound antibody by western blotting.
10. Make small aliquots and try snap freezing. If antibody still works after thawing,
make aliquots and snap freeze them. Antibody should not be freeze-and-thawed
repeatedly, so make aliquots small enough. Store at –20ºC with 50% glycerol.
* Eluting with 100mM glycine better preserve antibody active so that it is recommended
for the first attempt. However, if the purpose of affinity purification is for
immunostaining and the purified antibody doesn’t give good staining, then alternative is
to use 3.5M MgCl2 in PBS for elution. Use of 3.5M MgCl2 give better chance of eluting
high affinity antibody from the column, but it is very harsh to both antibody and column.
Both dialyzing elutant and washing column should be done immediately.
Kyoko Okada, 02/21/2005