Overview

  • Product name
    Antibody Serum Purification Kit (Protein A)
    See all Antibody Purification kits
  • Product overview

    Antibody Serum Purification kit (ab109209) is prepared by coupling highly purified protein A to agarose beads and can therefore be used to purify IgG fractions from both serum and ascites fluid.


    The antibody is captured on the resin and unwanted substances are removed by a simple wash procedure. The purified product is then eluted and neutralized.


    The components of ab109209 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).


     


    ab109209 is not suitable for goat antibody purification.

  • Notes

    ab109209 contains 1-3 columns, and each can purify up to 20mg of antibody per run.

    The volume of sample required will depend on the host species (See Table below):

     

    Species Normal range IgG(mg/ml) Suitable Volumes for Product(ml)
    Rabbit 12 - 15 1.3 - 1.7
    Human 7 - 23 0.9 - 2.9
    Mouse 2 - 5 4 - 10
    Sheep/Goat 18 - 24 0.8 - 1.1
    Rat 5 - 7 2.9 - 4
    Ascites Fluid 0.5 - 5 4 - 40

     

    Protocol

    1. Serum or Ascites Fluid preparation

    Add the 10x Binding buffer to the serum or ascites fluid (add 1/10 of the volume of sample). For example, for 5ml of serum add 0.5ml of 10x Binding Buffer and mix by inversion.

    NOTE: for samples volumes of less than 5ml, dilute the sample with wash buffer to 5ml before adding the 10x Binding Buffer.

    2. Incubate the sample with resin

    Add the protein A resin to the prepared sample and incubate with mixing at RT for a minimum of 2 hours. Alternatively, incubate overnight at either 4°C or room temperature. Use the sample to rinse the glass vial to recover all protein A resin.

    Please note that protein A resin has less affinitiy for sheep antibodies than for mouse/rabbit antibodies, and this will affect the binding capacity.

    3. Packing of the column

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

    Unwanted supernatant will pass through the column and can be kept on ice until a successful outcome has been confirmed.

    4. Wash procedure

    Wash the column with 7ml of Washing buffer to remove any non-bound protein. Repeat the washing step three times.

    NOTE: wash the inner surface of the column to remove any residual starting material.

    5. Elution

    Note: Elute the antibody in 1ml fractions.

    1. Place a set of collection tubes under the column ready for elution. Add 1ml of Elution Buffer to the column and collect through-out liquid.
    • Remove the collection tube from underneath the column and add 250µl of Neutralizing 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.

     

    The Neutralizing buffer must be added as soon as possible to the sample to avoid prolonged expossure to low pH which can result in denaturation of the IgG.

    The IgG normally elutes in Tubes 1 and 2 but you should confirm this using a test for protein before pooling any of the tubes.

    Antibody Concentration (optional)

    If the concentration of the recovered antibody is low then it can very quickly and easily be concentrated using our Concentration kit (ab102778).

    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, as this can interfere

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 3 tests 1 tests
    10x Binding Buffer 1 unit 1 unit
    Elution Buffer 1 unit 1 unit
    Neutralizer 1 unit 1 unit
    Protein A resin 3 units 1 unit
    Purification Column 3 units 1 unit
    Wash Buffer 1 unit 1 unit
  • Research areas

Protocols

References

This product has been referenced in:
  • Volgers C  et al. Budesonide, fluticasone propionate, and azithromycin do not modulate the membrane vesicle release by THP-1 macrophages and respiratory pathogens during macrophage infection. Inflammopharmacology 25:643-651 (2017). Read more (PubMed: 28528362) »
See 1 Publication for this product

Customer reviews and Q&As

1-4 of 4 Q&A

Answer

It is true that in the protocol we recommend using 1.3-1.7ml of rabbit IgG.

0.5ml volume is therefore too low to use with this purification kit, so we would recommend diluting the antibody in a binding buffer to get a volume of 1.3ml and continuing with the protocol as normal . We do not see any reason why diluting the antibody further would affect their downstream process’ as there is an antibody concentration step at the end the protocol.

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Answer

Thank you for taking the time to contact us. I am sorry to hear you have had difficulty obtaining satisfactory results from this purification kit.

I fully understand your concerns and it is regrettable the result have not been successful on this occasion. I am sorry to confirm that sheep antibodies do not have any affinity to protein A, so I would not expect this kit to work for sheep samples. In the previous email, advice was provided to confirm that the system for this kit is based on a Protein A purification and that the performance of the kit will therefore be both species and sub type dependent.

I would recommend a protein G kit would be more suitable for sheep antibodies, for example:

ab128751 Serum Antibody Purification Kit (Protein G)
https://www.abcam.com/index.html?datasheet=128751 (or use the following: https://www.abcam.com/index.html?datasheet=128751).

If you would like to try this kit instead, I can offer you a 5% discount and free shipping for this kit. Please quote the followingcodes:

5% discount: #####
Free shipping: ####

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

Read More

Answer

Thank you for your phone calls.

I have heard back from the lab and they recommend using the purification kit ab102784 for small volumes of tissue culture supernatant or ascites fluid (100-500 uL). When using this kit, it will be fine to dilute your 60 uL of ascites fluid to 100 uL with PBS.

The purified antibodies can then be used with our EasyLink conjugation kits. For optimal results, the concentration of the antibody should be between 0.5-5 mg/mL. You may still be able to conjugate the antibody at lower concentrations, but it will likely not be as efficient and may result in unlabeled antibody present in the solution.

If you switch to the purification kits for smaller volumes, the concentration will likely not be a problem, but if you dilute your antibodies for use with the TCS or Ascites purification kits, you may need to concentrate it before use with the EasyLink kits. This can be done by using our concentration spin columns ab102776 or ab102778.

Since you have already purchased the TCS and Ascites purification kits, I will be happy to arrange an exchange if you would like to switch to the kits designed for smaller volumes. Let me know if you need any other information or assistance.

Read More

Answer

Thank you for your enquiry,

I can confirm that the testing oftheab109209 Antibody Serum Purification kit has been done on serum samples. Although there is no obvious reason why the kits should not work on plasma, I am sorry we presently have no data to confirm and guarantee this.

I would also advise that the system is based on a Protein A purification. The performance of the kit will therefore be both species and sub type dependent.

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

Read More

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