Methods and materials:
Running affinity columns
Affinity
purification of antibodies
Running
an affinity column.
To read
about how to make a column from bacterial protein or peptides jump to the making
a column page
Important
information on elution conditions!!!
Different antibodies are eluted off affinity columns under different conditions.
Most will come off in the glycine elution protocol outlined here. You can test
if they came off or not by applying the "eluted" antibody to a small piece of
nitrocellulose that has a spot of your protein on it and developing it by normal
protocols. Glycine is the best option, as not only does it work with most antibodies
it also causes less damage to the column itself. If it doesn't come off with
glycine, try the ethanolamine base protocol, which is also not too rough of
the column. If nothing comes off with either of these, try the high magnesium
elution, while this will probably work it may (=probably) also destroy your
column.
Preparation
of serum
- Thaw
serum at room temp.
- Place
in 56 degC water bath for 30' to inactivate complement.
- Cool
on ice.
- Spin
in eppendorf for 2' to remove scum, transfer clear serum to clean tube and
store on ice.
Preparation
of column for first use.
Except where
noted, perform this entire procedure at 4 degC
The column should be prewashed with each of the solutions that will be used
in the purification to ensure that any materials that will be eluted/removed
by them do not do so during the purification. For example two different elution
buffers will be used and you want to make sure the acidic or basic solutions
wash off anything they can before we add the serum. If you have tested your
antibody and found it comes off well in glycine, skip all of the basic etahanolamine
steps The column bed volume is probably 0.5 to 1.0 ml.
- Wash
with 5 ml of TBS (recipes), allow to drain.
- Wash
with 20 mM Tris pH 7.5 500 mM NaCl, drain.
- Wash
with 5 ml of glycine (recipes), allow to drain
- wash
with 5 ml of 50 mM Tris pH 8.8, allow to drain.
- Wash
with 5 ml Ethanolamine (recipes), allow to drain.
Wash with
2 x 5 ml TBS. Ready to use.
Subsequent runs can be made by pre-washing with TBS.
Affinity
purification
Before you
start
prepare
10 eppendorf tubes each containing 42 ul of 1M Tris Base and 50 ul 5 mg/ml BSA.
Label these tubes with the antisera number and Gly#1 to Gly#10, these will be
used for glycine elution.
Prepare
another set of 10 tubes containing 50 ul of 1M Tris pH7.5 and 50 ul 5 mg/ml
BSA.Label these tubes eth#1 to eth#10. These will be used to collect ethanolamine
eluate.
- dilute
serum 1:10 with TBS, apply to column.
- Collect
the serum and pass over the column again. Collect and pass over the column
a third time. Collect serum and store at 4degrees C.
- wash
column with 2 x 5 ml of TBS
- wash
column with 2 x 5 ml 20 mM Tris pH7.5, 500 mM NaCl
- wash
column with 10 x 0.5 ml of glycine, collect 500 ul aliquots into the prelabelled
tubes containing Tris base and BSA. Add the first 0.5 ml, allow it to run
through and collect it, then add the next 500 ul, etc.
- wash
column with 5 ml of glycine. If you know your antibody comes off in glycine
skip to step 13.
- wash
column with 5 ml of 50 mM Tris pH 8.8.
- wash
column with 10 x 0.5 ml of ethanolamine pH 11.5. Collect each fraction into
prelabelled tubes containing tris and BSA
- wash
column with 5 ml of ethanolamine
- wash
with 10 x 0.5 ml 3.5 M MgCl2, collect aliquots.
- wash
column with 10 ml TBS.
- Store
all samples at 4 degrees C temporarily. Add azide to 0.1% for long term storage
in fridge.
- Check
antibody on wholemounts or nitrocellulose spot blots. Try 1/20 and 1/100 dilutions.
Label with details and freeze good aliquots.
Solutions
- TBS;
20 mM Tris pH 7.5 , 150 mM NaCl
- Tris/high
salt; 20 mM Tris pH 7.7, 500 mM NaCl
- Glycine;
100 mM Glycine pH 2.5 for acid elution
- 50 mM
Tris pH 8.8 (8.6 is OK)
- 100 mM
ethanolamine pH 11.5 for base elution
- 3.5 M
MgCl2, 20 mM Tris pH 7.5 for Mg elution
protocol
by Peter Vize
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