Overview
- Product nameAntibody Purification Kit (Protein A)See all Purification kits ...
- Product overview
Commercially available antibodies often contain substances (e.g. BSA, glycine, tris, azide) that interfere in labeling reactions with enzymes or fluorophores. Ab102784 quickly removes these contaminants. It can also be used to purify antibodies from crude samples such as ascites fluid or immune serum. The antibody to be purified or cleaned up ideally is in a volume of 100µl to 0.5ml. Up to 500µg of antibody can be purified in each run.
The method involves capture of the antibody on protein A resin and the removal of unwanted substances by a simple wash procedure, which is carried out in a standard microfuge. The purified product is then eluted and neutralized. Note: This method cannot be used with samples containing relatively dilute antibody in large volumes (e.g. tissue culture supernatant).For larger volumes, we recommend the use of Antibody TCS Purification Kit (3 purifications) (ab109207) or Antibody Serum Purification kit (3 purifications) (ab109209).
The components of Ab102784 are fully compatible with our EasyLink antibody conjugation kits.
ab102784 is not suitable for goat antibody pufication.
- Notes
Protocol
1. Reconstution of Protein A Resin
Add 0.5ml of wash buffer, mix by inversion for a few seconds and transfer to the spin cartridge. Spin for 30 seconds in a microfuge.
2. Incubation of Sample with Resin
To the antibody, add an appropriate amount of 10x Binding Buffer. For example, if the sample volume is 200µl, add 20µl of Binding Buffer. Pipette the sample into the spin cartridge and cap the tube. Incubate for 1 hour with agitation, end-over-end mixing or periodic shaking.
Note: The volume of antibody to be purified or cleaned up ideally should be 0.1-0.5ml, though larger volumes may be processed by first incubating the antibody sample with the protein A resin in a larger vessel (e.g. 2ml tube) prior to transferring to the spin cartridge.
Please note that protein A resin has less affinitiy for sheep antibodies than for mouse/rabbit antibodies, and this will affect the binding capacity.
3. Wash Procedure
Microfuge the spin cartridge assembly for 30 seconds to remove most of the non-bound protein. Add 0.5ml of wash buffer and spin again. Repeat the wash procedure three times.
Note: Save the non-bound and wash fractions by transferring the material from the collecting tube after each spin to a set of eppendorfs (not supplied). Do not use the four collecting tubes supplied with the kit, as these have an extended hinge to accommodate the spin cartridge, and are required for the elution step.
4. Elution
- Transfer the cartridge to a clean collecting tube. Add 100µl of elution buffer and incubate for 2 min at room temperature with gentle agitation. Microfuge for 30 seconds. Remove the collecting tube and add 25ul neutralizer to the tube.
- Place the cartridge in a new collecting tube and add a further 100µl of elution buffer to the protein A resin. Incubate for 2 min at room temperature with gentle agitation. Spin and collect and neutralize as before.
- Repeat the elution procedure until all four clean collecting cups have been used. The protein normally elutes in tubes 1 and 2 but you should confirm this using a test for protein (see note 3) before pooling any of the tubes.
- Pool the tubes with most protein (normally two tubes; if more than two tubes are strongly positive it is possible that you have used too much sample in your protein assay). However, if your application does not require a high concentration of antibody you may choose to pool all tubes that contain protein, regardless of concentration.
Storage of Antibody
Store at 4°C. Other storage conditions (e.g. frozen at -70°C may also be satisfactory). The sensitivity of any particular antibody to freeze thaw should be determined by experimentation on small aliquots.
Test for Protein
Wherever possible protein values should be determined using an absorbance at 280nm.
When other methods are used such as BCA or Bradford protein assays, determinations should be performed before the addition of the neutralization buffer. The neutralization buffer contains components that can interfere with these reagents. The neutralization buffer should be added to the sample as soon as possible as the low pH of the elution buffer can denature the antibody.
When using Bradford type reagents it is important to use an IgG standard curve. The absorbance generated by this type of reagent is dependent on the protein used. For example using a BSA standard curve to determine the protein concentration of an IgG solution will result in a two fold under estimate of the IgG concentration.
Properties
- Storage instructionsStore at +4°C. Please refer to protocols.
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Components Identifier 3 tests 1 tests 10x Binding Buffer 1 unit 1 unit Additional Collecting Tubes 4 units 1 unit Elution Buffer 1 unit 1 unit Lyophilised protein A resin White cap 3 vials 1 vial Neutralizer 1 vial 1 vial Spin Cartridge/ Collecting tube assembly 1 unit 1 unit Wash Buffer 1 unit 1 unit - Research Areas
References for Antibody Purification Kit (Protein A) (ab102784)
ab102784 has not yet been referenced specifically in any publications.
