Affinity purification of antibodies

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==Abstract==
==Abstract==
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This protocol describes purification of a polyclonal antibody that might be needed for immuno fluorescens microscopy or co-immuno precipitations.  
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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.
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'''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.
==Materials==
==Materials==
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==Procedure==
==Procedure==
===Coupling to activated beads===
===Coupling to activated beads===
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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 ([http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/C9142 sigma-aldrich])
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The first step is to couple the antigen to activated beads. In the following procedure CNBr-activated beads from Sigma are used and the procedure is modified from their product information ([http://www.sigmaaldrich.com/catalog/search/ProductDetail/SIGMA/C9142 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.
#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 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.

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Contents

Curators

Abstract

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.

Materials

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.

Reagents

Biological resources e.g. cell lines, buffers (link to a method for making them), enzymes, chemicals, kits, etc.

Equipment

Any equipment used to perform the protocol (link to a method for using them).

Procedure

Coupling to activated beads

The first step is to couple the antigen to activated beads. In the following procedure CNBr-activated beads from Sigma are used and the procedure is modified from their product information (sigma-aldrich)

  1. 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.
  2. 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.
  3. 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!
  4. 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).
  5. Wash away unreacted ligand using NaHCO3/NaCl coupling buffer described above.
  6. 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.
  7. 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).
  8. Complete this wash cycle of high and low pH buffer solutions four or five times.
  9. 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.

Critical steps

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Troubleshooting

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Notes

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Specific Protocols

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