Overview

  • Product name
    Antibody Purification Kit (Protein G)
    See all Antibody 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. ab128747 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 300µg of antibody can be purified in each run.


    The method involves capturing the antibody on the 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. Up to 300µg of antibody can be purified in each run. The purified product is then eluted and neutralized.


     


    The components of ab128747 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 our Gold antibody purification kit (ab204909).

  • Notes

    Protocol

    1. Reconstitution of Protein G Resin

    Add 0.3ml 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 a minimum of 2 hours with agitation or periodic shaking. Alternatively, incubate overnight at either 4°C or room temperature.

    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 G resin in a larger vessel (e.g. 2ml tube) prior to transferring to the spin cartridge.

    3. Wash Procedure

    Microfuge the spin cartridge assembly for 30 seconds at 13,000g 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 mins at room temperature with gentle agitation. Microfuge for 30 seconds at 13,000g. Remove the collecting tube and add 25µl neutralizer to the tube.
    • Place the cartridge in a new collecting tube and add a further 100µl of elution buffer to the protein G resin. Incubate for 2 mins 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 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 -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
    10 X Binding Buffer 1 vial 1 vial
    Collecting Tubes 4 units 12 units
    Elution Buffer 1 vial 1 vial
    Neutralizer Buffer 1 vial 1 vial
    Protein G Resin 1 vial 3 vials
    Spin Cartridges/Collection Tubes 1 unit 3 units
    Wash Buffer 1 vial 1 vial
  • Research areas

Protocols

References

ab128747 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-4 of 4 Abreviews or Q&A

Answer

The antibody purification kits that we carry, such as ab128745 and ab128747, will only recognize and purify IgG. For other classes such as IgA, IgD, and IgM, we have class-specific antibodies, for instance ab124716, which could be used to immunprecipitate those classes of immunoglobulin. Please let me know if you have any questions.

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Answer


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 0.01% sodium azide will inhibit HRP activity, and as you mentioned the 50% glycerol is making it hard to remove the sodium azide.

Antibody purification kit if you are going to stay with ab76424:
Protein A for rabbit antibodies ab102784: https://www.abcam.com/Antibody-Purification-Kit-Protein-A-ab102784.html
Protein G for other species ab 128747: https://www.abcam.com/Antibody-Purification-Kit-Protein-G-ab128747.html
For mouse antibodies ab128745: https://www.abcam.com/Mouse-Antibody-Purification-Kit-ab128745.html

Previous customers have contacted us with issues purifying antibodies with high concentrations of glycerol, and we found the following solution proved successful:
" When using the antibody purification kit the presence of glycerol in the antibody buffer will make it more viscous. This means that it will require more G force to pass through the spin column. It is possible to buffer exchange the antibody it just means the customer will have to spin for much longer. Alternatively, a simpler solution would be to dilute the antibody. This would reduce the glycerol’s concentration, which in turn will make the buffer less viscous and easier to spin out. You can then use the same spin column to concentrate the antibody back to its original concentration and volume, after having performed the buffer exchange."

Additionally, we have our EasyLink conjugation kits, including HRP conjugation, listed through the link below. Our EasyLink conjugation kits are capable of fully successful conjugation with glycerol concentrations of up to 50%. We are currently running a promotion for an extra 20% off our EasyLink kits:
EasyLink Kits: https://www.abcam.com/index.html?pageconfig=resource&rid=13148#enzymatic
Easylink Promotion:https://www.abcam.com/index.html?pageconfig=resource&rid=15389&source=pagetrap&viapagetrap=easylink123
Below is a list of compatible and incompatible buffers with our EasyLink conjugation reactions:
https://www.abcam.com/index.html?pageconfig=resource&rid=13156#11

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Answer

Thank you for contacting us.

I have been in contact with the originator of these products. Unfortunately they do not have any direct experience doing these types of procedures with pre-conjugated products and cannot provide any further information.

My colleagues and I believe that these procedures should be effective for removal of unwanted buffer additives from the antibody however we would likely recommend using the antibody concentration kit as it seems less likely that damage may occur. Our concentration kits may be found at:

1 column:

https://www.abcam.com/Antibody-Concentration-Kit-1-column-ab102776.html

3 columns:

https://www.abcam.com/Antibody-Concentration-Kit-3-columns-ab102778.html

Please remember that these products are covered by our Abpromise guarantee. We are happy to provide scientific support, replacement or refund should a product not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise

I hope that this information is helpful. Please let me know if you have any questions or there are other ways that Abcam may help you meet your research goals.

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Answer

Thank you for contacting us.

We currently do not offer ab8580 in a buffer which is free of BSA. When using our EasyLink Conjugation Kits concentrations of up to 0.5% BSA and 0.1% gelatin have little effect on the conjugation chemistry. As such I would suggest one of the following options.

1) Purify the antibody to remove the BSA and Sodium Azide. We carry antibody purification kits which canquickly remove these contaminants. Our Protein G purification kit may be found here:https://www.abcam.com/Antibody-Purification-Kit-Protein-G-ab128747.html

2) Use another anti Histone H3 K4me3 antibody which does not contain BSA or other additive at levels which will interfere with your conjugation.AB12209 contains only 0.2% Sodium Azide and no BSA.https://www.abcam.com/Histone-H3-tri-methyl-K4-antibody-mAbcam12209-ChIP-Grade-ab12209.html

More information and FAQs about EasyLink Conjugation Kits may be found at these links:

FAQs: https://www.abcam.com/index.html?pageconfig=resource&rid=13156

EasyLink:https://www.abcam.com/index.html?pageconfig=resource&rid=13148

We are currently running a promotion through August for 25% off of any EasyLink conjugation kit by using the codeNEW-F76ZBby 31 August 2012.

As always, these products are covered by our Abpromise guarantee. We provide scientific support, replacement or refund should this product not perform as indicated on the datasheet. More information on our Abpromise may be found at the following link:

https://www.abcam.com/index.html?pageconfig=abpromise


I hope this information is helpful to you. Please do not hesitate to contact us if you need any more advice or information.

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