Properties for AbSelect G Antibody Purification System
|Product Category||Labeling Kits & Reagents|
|PDF datasheet||View Datasheet|
|Manufacturer||Acris Antibodies GmbH|
|Background||Commercially available antibodies often contain substances (e.g. BSA, Glycine, Tris and Azide) that interfere in labeling reactions with enzymes, Biotin, Streptavidin or Fluorophores. The AbSelect G Purification System AC053 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 0.1 ml to 0.5 ml. Up to 500 ug of antibody can be purified in each run.
The method involves capturing the antibody on the AbSelect Protein G Resin. Protein G has a high affinity for the Fc regions of IgG molecules from a variety of species. Once the antibody has bound to the Protein G, unwanted substances can be removed by simply washing the resin. The purified product is then eluted and neutralized.
The AbSelect G Purification System is fully compatible with both the Light-A-Link conjugation systems (available separately), which allow the purified antibody to be immediately labeled with a hands-on time of under 30 seconds.
|Protocols||1. Reconstitution of AbSelect Mouse Resin
Add 0.3 ml of Wash Buffer AC053-003-W to each vial of Abselect Mouse Resin, mix by inversion for a few seconds and transfer to the Spin cartridge AC053-003-T1 (Figure 1). Spin for 30 seconds in a microfuge.
2. Incubation of Sample with Resin
Add the appropriate amount of 10x Binding Buffer AC053-003-B to the antibody. For example, if the sample volume is 200 µl, add 20 µl of Binding Buffer. Pipette the sample into the spin cartridge and cap the tube. Incubate for 1 hour with agitation or periodic shaking.
Note: The volume of antibody to be purified or cleaned up should be 0.1-0.5 ml, though larger volumes may be processed by first incubating the antibody sample with the Protein A Resin AC053-003-R in a larger vessel (e.g. 2ml eppendorf) prior to transferring to the spin cartridge.
3. Wash Procedure
Microfuge the spin cartridge assembly for 30 seconds to remove most of the non-bound protein. Add 0.5 ml of Wash Buffer AC053-003-B and spin again. Perform 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 AC053-003-T2 supplied with the kit, as these have an extended hinge to accommodate the spin cartridge, and are required for the Elution step.
Transfer the cartridge to a clean collecting tube AC053-003-T2. Add 100 µl of Elution Buffer AC053-003-E and incubate for 2 minutes at room temperature with gentle agitation. Microfuge for 30 seconds. Remove the collecting tube (See Section 5) and add 25 µl Neutralization Buffer AC053-003-N.
Place the cartridge in a new collecting tube and add a further 100 µl of Elution Buffer AC053-003-B to the Protein A Resin. Incubate for 2 minutes at room temperature with gentle agitation. Spin and collect and neutralize as before.
Repeat the Elution procedure until all four clean collecting tubes have been used. The protein normally elutes in tubes 1 and 2 but you should confirm this using a test for protein (See Section 5) before pooling any of the tubes.
Pool the tubes with the most protein (this is 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.
5. 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 AC053-003-N.
The Neutralization Buffer AC053-003-N 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 AC053-003-E 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 2.3-fold under-estimate of the IgG concentration.
|Storage||Storage of Kit: The kit is shipped at ambient temperature. Store the kit at 4°C upon receipt.
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.