Overview

  • Product name
    Serum Antibody Purification Kit (Protein G)
    See all Antibody Purification kits
  • Sample type
    Serum
  • Product overview

    Protein G has a high affinity for the Fc regions of IgG molecules from a variety of species. ab128751 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. Up to 10mg of antibody can be purified in each run. The volume of sample required will depend on the host species.

    The purified product is then eluted and neutralized.

     

    The components of ab128751 are fully compatible with our Conjugation kits however they are not compatible with our GOLD Antibody conjugation kits. To purify antibodies for use with our GOLD conjugation kits, please use Gold antibody purification kit (ab204909).

     

     

     

  • Notes

    Protocol

    Overview of procedure

    Step 1: Prepare the serum or ascites fluid.

    Step 2: Transfer the Protein G resin to the prepared sample and mix for 2 hours.

    Step 3: Transfer the solution into the column.

    Step 4: Wash the Protein G resin.

    Step 5: Elute and neutralize purified antibody.

    Step 6: Confirm antibody is in eluate using a test for protein.

    Step 7: Concentrate the antibody (optional).

    1. Preparating the Serum or Ascites fluid

    Add the 10x Binding Buffer to the serum or ascites fluid. The volume to add is 1/10 of the volume of the sample. For example, for 5ml of serum add 0.5ml of the Binding Buffer. Mix by inversion.

    Note: For sample volumes of less than 5ml, dilute the sample with Wash Buffer to 5ml before adding the Binding Buffer.

     

    2. Incubation of Sample with Resin

    Add the resin to the supernatant and incubate with mixing at room temperature for a minimum of 2 hours. Use the sample to rinse the bottle to recover all the resin.

     

    3. Packing of the column

    Carefully pour the sample-resin mix into the column. Sample volumes of more than 10ml will have to be added in aliquots. The 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.

    Note: Up to 10mg of antibody can be purified in each run. The volume of sample required will depend on the host species.

    4. Wash procedure

    Wash the column with 7ml of Wash Buffer 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.

    5. Elution

    The antibody is eluted in 1ml fractions. Place a collecting tube under the column and add 1ml of Elution Buffer (see below). Remove the collection tube and add 0.25ml of Neutralization Buffer. Cap the tube and place to one side. Repeat the elution process three more times, each time neutralizing the sample as it is eluted.

    Note: The Neutralization Buffer 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 before pooling any of the tubes.

    Storage of Antibody

    Store at 4°C. Other storage conditions (e.g. frozen at -80°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 instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 tests 3 tests
    10x Binding Buffer 1 vial 1 vial
    Elution Buffer 1 vial 1 vial
    Neutralizer Buffer 1 vial 1 vial
    Purification Column 1 unit 1 x 3 units
    Serum Protein G Resin 1 vial 1 x 3 vials
    Wash Buffer 1 vial 1 vial
  • Research areas

Protocols

References

ab128751 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-3 of 3 Q&A

Answer

I can confirm that a) an entire bottle of resin is to be used when purifying a single antibody with the 1x kit b) the 3 test kit has 3 separate vials of resin.

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Answer

Thank you for your enquiry. Here are the answers to the questions posed above:


question1): Does this kit (serum antibody purification kit (protein G)) - ab128751 work in hamster serum?
Yes. Protein G has moderate affinity for hamster IgG and should give results.

question 2:) If this works on hamster serum, how many kits would be necessary to purify 3-4 ml of hamster serum?
Normal hamster serum IgG levels are between 8 and 17mg/ml. This kit can bind up to 10mg of rabbit IgG therefore two to seven kits would be necessary.
question 3:) Are there any additional supplies or reagents necessary to run this kit, or with hamster?
No.

Please let us know if you have further questions.

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Answer

Thank you for your enquiry.

I can confirm that sheep antibodies do have an affinity to protein G, so the Serum Antibody Purification Kit (Protein G) (ab128751)" should be suitable forsheep serum IgG purification.

Further information on protein A and G purification and compatibility can be found on the followingdocument from our protocols page:

https://www.abcam.com/index.html?pageconfig=resource&rid=11385

I hope this will be helpful to you. If you have any further questions, please do not hesitate to contact us.

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