Properties for AbSelect G Serum Antibody Purification System
|Product Category||Labeling Kits & Reagents|
|Reaction||3 Purifications, each up to 10mg IgG|
|PDF datasheet||View Datasheet|
|Manufacturer||Acris Antibodies GmbH|
|Background||Antibodies generated from ascites fluid and serum are often supplied as crude formulations. Both ascites fluid and serum contain many substances that interfere with antibody labeling reactions. Successful labeling therefore requires antibodies to be purified prior to labeling. The AbSelect Serum System AC054 is a fast and simple method to purify antibodies from these types of media.
Protein G has a high affinity for the Fc regions of IgG molecules from a variety of species. AbSelect G Serum resin is prepared by coupling purified Protein G to agarose beads. It can therefore be used to purify IgG fractions from both serum and ascites fluid. The antibody is captured on the AbSelect G resin and unwanted substances are removed by a simple wash procedure. The antibody is then eluted and neutralized.
The components of the AbSelect Serum purification system are fully compatible with the Link-A-Light antibody labeling kits (available separately), which allows the purified antibody to be immediately labeled with an enzyme, fluorescent protein or fluorescent dye, with a hands-on time of under 30 seconds.
|Protocols||1. Amount of antibody that can be purified
Up to 10 mg of antibody can be purified in each run. The volume of sample required will depend on the host species (See Appendix 2).
2. Preparing the Serum or Ascites Fluid
Add the 10x Binding Buffer AC054-003-B to the Serum or Ascites fluid. The volume to add is 1/10 of the volume of the sample. For example, for 5 ml of Serum add 0.5 ml of 10x Binding Buffer AC054-003-B. Mix by inversion. Note: For sample volumes of less than 5 ml, dilute the sample with Wash Buffer AC054-003-W to 5 ml before adding the 10x Binding Buffer AC054-003-B.
3. Incubation of Sample with Resin
Add the AbSelect Resin AC054-003-R to the Supernatant and incubate with mixing at room temperature for a minimum of 2 hours. Use the Supernatant to rinse the bottle to recover all AbSelect Resin.
4. Packing of the Column
Carefully pour the Sample-Resin mix into the column. Sample volumes of more than 10 ml will have to be added in aliquots. The AbSelect Serum Resin will collect in the bottom of the column.
The unwanted Supernatant will pass through the column and can be kept on ice until a successful outcome has been confirmed.
5. Wash Procedure
Wash the column with 7 ml of Wash Buffer AC054-003-W to remove any non-bound protein. Repeat the wash procedure three times.
Note: Wash the inner surface of the column to remove any residual starting material.
The antibody is eluted in 1 ml fractions.
Place a collecting tube under the column and add 1 ml of Elution Buffer AC054-003-E (See Section 8).
Remove the collection tube and add 0.25 ml of Neutralization Buffer AC054-003-N.
Cap the tube and place to one side.
Repeat the elution process three more times, each time neutralizing the sample as it is eluted.
The Neutralizing Buffer AC054-003-N must be added as soon as possible to avoid prolonged exposure to low pH which can result in denaturation of the IgG.
The protein normally elutes in tubes 1 and 2 but you should confirm this using a Test for protein (Section 8) before pooling any of the tubes.
7. Antibody Concentration (Optional)
If the concentration of the recovered antibody is low then it can very quickly and easily be concentrated using our Antibody Concentration and Clean Up Kit (Cat.-N AC048 available separately).
8. Test for Protein
Wherever possible protein values should be determined using absorbance at 280nm.
When other methods of are used such as BCA or Bradford protein assays, determinations should be performed before the addition of the Neutralization Buffer AC054-003-N, as this can interfere with these reagents. Remove an aliquot for protein determination and neutralize the rest of the fraction immediately 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. Failure to do this will result in incorrect protein levels being calculated. If IgG is not available then a BSA standard curve can be used, but the IgG levels will be under-estimated by a factor of 2.3.
|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.