Overview

  • Product name
    Antibody Concentration Kit
  • Product overview

     


    Antibody Concentration Kit ab102778 allows for the quick and easy concentration of antibodies and proteins using a microcentrifuge-based method. The kit can also be used to reduce the concentration of many unwanted additives often found in antibody formulations such as azide, glycine or tris.


    Antibodies are sometimes only available at low concentrations and often contain low molecular weight substances that interfere in labeling reactions with enzymes, biotin, streptavidin and fluorophores. ab102778 can be used to avoid the impact of this.


    The Antibody Concentration Kit method utilizes a simple spin column to easily and quickly remove excess buffer from the antibody thereby providing a more concentrated antibody solution. The Antibody Concentration Kit also allows the experimenter to perform a simple buffer exchange to transfer the antibody.


    The components of Ab102778 are fully compatible with our Antibody conjugation kits however they are not compatible with our GOLD Antibody conjugation kits. To concentrate antibodies for use with our GOLD conjugation kits, please use our Gold antibody concentration kit (ab204911).


     

  • Notes

    Concentration of Antibody Solution

    1. Add antibody to spin cartridge.
    2. Spin for 1 to 3 minutes* in a microfuge at max speed of 15000g to reduce the buffer volume in the spin cartridge to between 50 and 100ul. It is advisable not to spin the antibody dry as reconstitution of the antibody will be difficult and significant antibody loss and degradation may occur.
    3. Repeat steps 1 to 2 as many times as is necessary to process the entire antibody to the desired concentration. It may be necessary to discard the excess buffer collected in the collection tube between spins.
    4. Recover the concentrated antibody from the spin cartridge. NB. It is advisable not spin the antibody dry as reconstitution of the antibody will be difficult and significant antibody loss and degradation may occur.

     Buffer Exchange Using Spin Column Assembly

    1. Add up to 0.5ml antibody to spin cartridge.
    2. Spin for 1 to 3 minutes* in a microfuge at maximum speed to reduce the buffer volume to 100ul.
    3. Discard the excess liquid in collection tube.
    4. Add 400ul conjugation buffer to the antibody in the spin cartridge.
    5. Spin for 1 to 3 minutes* in a microfuge at maximum speed to reduce buffer volume to 100ul.
    6. Discard the excess liquid in collection tube.
    7. Repeat steps 4 to 6 at least 5 times to exchange antibody buffer.
    8. Recover antibody from the spin cartridge.

     *Spin times will vary depending on buffer composition, starting volume and centrifuge speed.

    Note Each cycle leads to a reduction in the concentration of low molecular substances. By performing as many as 5 repeat steps the concentration of small molecules such as glycine and Tris will be reduced 2500 fold. However, the concentration of proteins such as BSA will be unchanged.

     To remove unwanted proteins see ab102784.

    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. For an IgG using a 1cm light path an OD280 of 1.0 is equal to an antibody concentration 0.714mg/ml.

    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 3 units
    Conjugation Buffer 1 unit
    Spin Cartridge/ Collecting tube assembly 3 units
  • Research areas

Images

Protocols

References

This product has been referenced in:
  • Röper JC  et al. The major ß-catenin/E-cadherin junctional binding site is a primary molecular mechano-transductor of differentiation in vivo. Elife 7:N/A (2018). Read more (PubMed: 30024850) »
See 1 Publication for this product

Customer reviews and Q&As

1-10 of 26 Q&A

Answer

Thank you for your enquiry.
The kit can be re-used up to 6 times. However, it can only be re-used with the same antibody. You could put wash buffer on its own through the column in between washes to clean it, but large particles will never get completely removed from the column so we would not recommend re-using the kit with other antibodies or proteins due to cross-contamination issues.
I hope this information will be helpful. If you have any further questions, please do not hesitate to contact me.

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Question
Answer

Thank you for your call and your patience while I communicated with my colleague in the lab.

In response to your inquiry, it turns out that both options (ab102784 Antibody Purification Kit (Protein A) or ab102778 Antibody Concentration Kit ) will work for your antibody purification purposes. However it turns out that the simplest kits would be the Concentration kit.

The spin columns provided in this kit allow a simple buffer exchange by ‘spinning away’ the existing buffer, and allowing this to be replaced by a more compatible buffer. As spinning the antibody dry is not best practice, as this can impact the antibody, the protocol states that this allows the azide to be reduced, as there will be a small amount of azide left in the little bit of buffer that remains. However if this process is repeated, each time, the amount of azide remaining will reduce more and more, therefore after enough spins, the azide concentration will effectively become zero. On the whole, this azide should be reduced to a suitable level almost after 1 buffer exchange, however we would recommend this is repeated further if the label of choice is HRP, as this has shown to be more sensitive. I hope you find this information helpful.

Please let me know if I can offer any more help or technical advice. Good luck with your research.

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Answer


The Antibody Concentration Kit method utilises a simple spin column to easily and quickly remove excess buffer from the antibody thereby providing a more concentrated antibody solution. Only molecules smaller than 10 kDa will be pass through the filter. Therefore, FITC alone will pass through the filter, unless it is conjugated to a molecule larger than 10 kDa, such as the antibody that you are concentrating.

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Answer

Most benchtop microcentrifuges have a maximum spin of 14,000 - 15,000 RPM, and a maximum RCF of 18,000 - 21,000 x g. Anywhere in that range will be fine. You might want to test spinning just buffer without antibody, to see how long it takes to reduce 500 ul (or less) to 100 ul, but an antibody solution will likely take longer, since the antibody blocks the filter. You can always check every 30 seconds to see how much the volume has been reduced, taking notes. After the first spin, the subsequent spins should take the same amount of time, assuming you add conjugation buffer after each spin (for example, PBS) to bring the volume up to the original volume of the Tris-buffered antibody solution.
The hydroxynonenal antibody ab48506 will be 50 ug in 500ul, a concentration of 0.1 mg/ml. You will want to concentrate the antibody with the same spin column by not adding more buffer after the last buffer exchange spin. Ideally, you want a concentration of at least 1.0 mg/ml for efficient conjugation.

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Answer

The maximum volume of antibody that you can add to the spin cartridge is 0.5 mL. However, you are can use the spin cartridge multiple times to concentrate the 3 mL antibody sample.

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Answer


The laboratory responded that the filter in the antibody concentration kit ab102778 should work fine with a 30KD fragment. It is possible that the filter could be damaged. It would help if you could measure the OD of the flow through. If the flow through has the same OD then there is a problem with the filter. If not, then there may be an issue with the OD measurements, perhaps a blanking issue or issue with the reader itself.

Please let me know the results of the OD readings of the flow through. I look forward to your reply.

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Question
Answer

Thank you for contacting us. We do not currently sell ab15272 without sodium azide, but we may be able to custom formulate the antibody for bulk orders of more than 10 vials. For enquiries about a custom formulation, please contact mailto:sales@abcam.com. For smaller quantities, you may be able to perform your own buffer exchange to significantly reduce the amount of sodium azide by using our concentration kits: ab102776 and ab102778. I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for contacting us. We may be able to custom make this antibody in an azide-free form for bulk orders of 10 or more vials. If you are interested in a custom bulk purchase, please contact my colleagues at mailto:sales@abcam.com and they can help put together a quote and estimated availability time for you.

Alternatively, for smaller purchases you could use one of our concentration and clean up kits (ab102778) to perform a buffer exchange and greatly reduce the concentration of azide in the antibody. I hope this helps, please let me know if you need any additional information or assistance.

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Answer

Thank you for contacting us regarding ab58555.

I have contacted the lab on your behalf, the antibody would be fine as well in a different buffer containing another stabilizing agent e.g. BSA. However this product is provided in PEG and sucrose as was found the antibody was suffering less during lyophilization.

The antibody itself is not stickier than other antibodies. It seems possible that the buffer with the PEG and sucrose is responsible of the low yield that you are getting after the conjugation. The buffer exchange method may need to be adjusted for improved performance.

We would recommend use of one of our concentration kits for performing buffer exchange with this product. Our antibody concentration and clean up kits, Ab102776 and ab102778utilizes a simple spin column to easily and quickly buffers. These should work in any buffer containing PEG and are covered by our Abpromise guarantee. Which guarantees scientific support, replacement or refund should any Abcam product not perform as indicated on the datasheet. More information about Ab102776, ab102778 and our Abpromise may be found at the link below.



Ab102776:https://www.abcam.com/Antibody-Concentration-Kit-1-column-ab102776.html

Ab102778:https://www.abcam.com/Antibody-Concentration-Kit-3-columns-ab102778.html

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 Abcam regarding our EasyLink conjugation kits.


I have reviewed the product specs of the antibody you are using and the buffer formulation is compatible with the HRP kit ab102891. As you mentioned, the only modification I would recommend is to further concentrate the antibody for optimal results. I would recommend ab102778 for this purpose, as you have already suggested. You should not require any additional reagents to successfully complete the conjugation reaction.


I hope this information is helpful. Please do not hesitate to contact me if you have any additional questions.

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1-10 of 26 Q&A

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