Antibody Purification Kit (Protein A) (ab102784)

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  is ideally in a volume of 100-500µl. 

    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 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 at room temperature, 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.

    Incubation at 4°C will significantly reduce the performance of the kit. If you wish to incubate the antibody with the resin at 4°C then you should extend the incubation step to overnight.

    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

    1. 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.
  • 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
    Lyophilized 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

    Protocols

    References for Antibody Purification Kit (Protein A) (ab102784)

    ab102784 has not yet been referenced specifically in any publications.

    Product Wall



    In cases where the antibody volume is larger, i.e. in your case where you have 1ml of antibody, we recommend you do the following:



    First, add the binding buffer to your antibody. Then add the binding buffer and antibody mix...

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    Protein values should be determined using an absorbance at 280nm. I have checked with our scientists, and they informed me that an absorbance (or OD) of 1 at 280nm corresponds to an antibody concentration of 0.1714mg/ml. Please note this value is ...

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    Thank you for contacting us.

    I am afraid that this kit doesn’t work for purification of IgMs. IgMs are normally purified by precipitation.

    I hope this information is helpful to you. Please do not hesitate to contact us if you ne...

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    As we discussed, our studies have found little effect on the conjugation chemistry with up to 0.05% BSA and up to 0.1% glycine in the antibody storage buffer. Thus, I think you do not have to worry about those components. However, as you know the ...

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    This kit will certainly suit your needs for removing the Tris-glycine and glycerol from your antibody before use with the EasyLink conjugation kit. While the EasyLink kits are able to be used with as much as 50% glycerol content they do need Tris level...

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    Thank you for your enquiry.

    We would recommend to aim for a maximum of 15,000g. Lower speeds will also be fine, it just may require slightly longer spin times to achieve the objective.

    I hope this will help. If you require any fur...

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    Thank you once again for your reply and patience.

    I am pleased to now have further information for you. We would recommend to use the following steps in this case:

    Add the resin to the 1 ml of antibody in a 1.5 ml eppendorf. Then ...

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    Thank you for contacting Abcam.

    Please find the link to the antibody purification kit below, that will allow you to remove the sodium azide from your antibody solution:

    http://www.abcam.com/antibody-purification-kit-protein-a-ab1027...

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    Thank you for bringing this to our attention. Assuming you followed the incubation and elution protocol, I am not sure what could have gone wrong. We have not had any complaints in the past several months. Did you try getting an absorbance reading for ...

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    Thank you for contacting us.

    I have checked with the lab and they sent me the following explanation:

    TheElution Buffer is used to elute (release) the purified IgG from the Protein A resin. The Elution Buffer works by low pH, and is...

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    Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"