Affinity purification of antibodies
- Torsten Waldminghaus 09:31, 3 November 2008 (EST):
This protocol describes purification of a polyclonal antibody that might be needed for immuno fluorescens microscopy or co-immuno precipitations. A comman way to do this is to couple the antigen to activated beads on a column, run the serum over it and than after some washing elute the antibody that was bound to the antigen. However this protocol describes a kind of 'affinity substraction'. Instead of the antigen a protein extract of an antigen deletion strain is coupled to the beads. The advantages are that one does not need purified antigen and that one can simply use the flow through instead of having to elute and dialyze. The disadvantage off course is that one needs a deletion strain which might be difficult if the respective protein is essential.
Note:This protocol is currently under developement and testing in the lab. So please watch changes over the next weeks and edit and comment all that you can.
List everything necessary to perform the protocol here. Include all information about suppliers, ordering details, etc. Links to the suppliers' page on that material are also appropriate and encouraged. Please be aware that users of this protocol may not be working in the same country as you.
Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.
Any equipment used to perform the protocol (link to a method for using them).
Coupling to activated beads
The first step is to couple the antiben to activated beads. In the following procedure CNBr-activated beads from Sigma are used and the procedure is taken from there Product information (sigma-aldrich)
- Dissolve protein to be coupled in 0.1 M NaHCO3 buffer containing 0.5 M NaCl, pH 8.3-8.5 (about 5-10 mg protein per mL of gel). Note: Other buffers can be used, but avoid amine-containing buffers such as Trizma or other nucleophiles (buffers with amino groups) which will react with the binding sites.
- Wash and swell cyanogen-bromide activated resin in cold 1 mM HCl for at least 30 minutes. A total of 200 ml per gram of dry gel is added in several aliquots. Remove the supernatant (contains lactose) by gentle suction in a Büchner or suction funnel between successive additions. Note: Lactose is necessary to stabilize the beads during freeze-drying, but it will interfere with binding if present during coupling. The use of HCl preserves the activity of the reactive groups which hydrolyze at high pH.
- Wash the resin with distilled water, 5 - 10 column volumes, then wash the resin with the NaHCO3/NaCl coupling buffer (5 ml per gram dry gel) and immediately transfer to a solution of the ligand in coupling buffer. Note: The reactive groups hydrolyze in basic solution!
- Mix protein with gel for 2 hours at room temperature or overnight at 4°C. Use a paddle stirrer or end-over-end mixer, but not a magnetic stir bar (which may grind beads).
- Wash away unreacted ligand using NaHCO3/NaCl coupling buffer described above.
- Block unreacted groups with either 1 M ethanolamine or 0.2 M glycine, pH 8.0 for 2 hours at room temperature or 16 hours at 4°C.
- Wash extensively to remove the blocking solution, first with basic coupling buffer at pH ≈ 8.5, then with acetate buffer (0.1 M, pH 4) containing NaCl (0.5 M).
- Complete this wash cycle of high and low pH buffer solutions four or five times.
- If the resin is to be used immediately, equilibrate it in buffer. If not, store the resin in 1.0 M NaCl at 2-8°C with a suitable bacteriostat.
Referenced from the main protocol, a more thorough explanation of particularly important steps in the protocol.
Referenced from the main protocol, an explanation of what can cause things to go wrong with the protocol.
Referenced from the main protocol, any comments about the protocol should be made here; i.e. how it was developed. Any comments added should be signed (by adding *'''~~~~''': in front) and explained. Links to FAQs/tips provided by other sources such as the manufacturer or other websites would be best made here.
Anecdotal observations that might be of use to others can also be posted here; e.g. 'my cells were still floating'.
It might also be good to add an image to show the workflow and timescales for experiment planning.
Acnkowledge any help you had in development, testing, writing this protocol.
See OpenWetWare:Biblio for information on how to reference within a wiki.
Add links to all the OWW protocols that have been used in making the consensus.
You can discuss this protocol.
Tag this page with categories to allow easier indexing and searching. See Categories for information on existing categories.