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.
- 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