Overview

  • Product name
    Antibody TCS Purification Kit
    See all Antibody Purification kits
  • Product overview

    Antibody TCS (Tissue Culture Supernatant) Purification kit has been designed for tissue culture supernant purification, and can be used to purify up to 50ml in each purification.

    This kit works by coupling highly purified protein A to agarose beads and can therefore be used to purify IgG fractions from hybridoma supernatants.The antibody is captured on the TCS resin and unwanted substances are removed by a simple wash procedure. The purified product is then eluted and neutralized.

    The components of this kit 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).

     

    ab109207 is not suitable for goat antibody purification.

  • Notes

    Amount and volume of antibody

    The antibody to be purified should be in 10 - 50ml of tissue culture supernatant.

    Each column can purify up to 5mg of antibody.

     

    Protocol

    1. Tissue culture supernatant preparation

    Add the 10x Binding buffer to the tissue culture supernatant (add 1/10 of the volume of tissue culture supernatant). For example, for 50ml of tissue culture supernatant add 5ml of 10x Binding Buffer and mix by inversion.

    2. Incubate the sample with resin

    Add the protein A resin to the prepared supernatant and incubate with mixing at RT for a minimum of 2 hours. Use the supernatant 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 supernatant-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.

    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 the antibody concentrator.

    1. Add antibody to the top of the spin cartridge.
    • Spin for 1 - 3 minutes in a microfuge at maximum speed to reduce the buffer volume in the spin cartridge to 50 - 100µl. (Spin times will vary depending on buffer composition and volume as well as centrifuge speed).

     

    • Repeat steps 1 and 2 as many times as is necessary to process the entire antibody to the desired concentration. It may be necessary to discard any excess buffer collected in the collection tube between spins.

     

    • Recover the concentrated antibody from the top of the spin cartridge.

     

    Note: It is advisable not spin the antibody dry as reconstitution of the antibody will be difficult and significant antibody loss and/or denaturation may occur.

    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 with these reagents. Remove an aliquot for protein determination and neutralize

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 3 tests 1 tests
    10x Binding Buffer 1 unit 1 unit
    Concentration Spin columns and Collection Tubes 3 units 1 unit
    Elution Buffer 1 unit 1 unit
    Neutralizer 1 unit 1 unit
    Purification Column 3 units 1 unit
    TCS protein A resin 3 units 1 unit
    Wash Buffer 1 unit 1 unit
  • Research areas

Protocols

References

ab109207 has not yet been referenced specifically in any publications.

Customer reviews and Q&As

1-7 of 7 Abreviews or Q&A

Answer

Thank you for contacting us. For a purified antibody (such as the one you are trying to purify) you would want to use either ab102783 or ab102784. They are exactly the same, except the first is for one purification and the second is forthree purifications, so depending on whether you may need to purify in the future you can choose which size is more suitable for you.

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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Answer

We have kits available for purifying antibodies from Tissue culture supernatants. The catalogue number is ab109207. Please check the datasheet; https://www.abcam.com/Antibody-TCS-Purification-kit-3-purifications-ab109207.html

1 test kit can cost €245 while 3 tests kit will cost €520. One kit will be enough to purify antibody from 10-50 ml of supernatant.

Please note if the antibody to be purified is in serum or other samples then kit ab109209 or ab102784 will be more suitable.

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Answer

Thank you very much for your email.
I am very sorry for this embarrassing error. We obviously should not have sent you this kit. I am very sorry again. Please accept my apologies for this error.
We do not have a purification kit specifically for tissue culture supernatant for small volumes. If you would like however, I can send you a free kit based on protein G. Indeed, we still do not know why the first kit, based on protein A, did not work. It might be just a problem of the kit/antibody or maybe this specific clone is not as good with protein A. It is up to you - I can either send you the protein G based kit ab128747 or again a kit of ab102784.
Please let me know how you would like to proceed. Again, all my apologies for this inconvenience.

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Answer

Thank you for your call last week and for your patience while I have been in touch with the lab regarding your enquiry.

I've heard back from the lab, and unfortunately this purification kit will not work with a small volume of tissue culture supernatant antibody. If you would be interested in ordering a larger quantity (10 or more vials) then please contact mailto:sales@abcam.com to see if we may be able to provide this antibody in a purified format.

Please let me know if you have any further questions or if there is anything else that we can do for you. Have a great day!

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Question

First issue: thank you for the offer: we would indeed appreciate a lot to receive a new vial of ab110363 and a purification kit for tissue culture supernatant ab109207 to replace the lost material. This could help us a lot.
Second issue: Questionnaire for ab21682:
IHC Questionnaire
It will take approximately 5 minutes to complete the questionnaire.
Please fill-in all fields so that we can better assist you
1) Abcam product code ab21682, rabbit anti-DLK1

2) Abcam order reference number or product batch number : GR31847-1
3) Description of the problem: high aspecific background, impossible/difficult to pick up the DLK1 signal. Conclusion: antibody is not specific, but gives a very particular intracellular/perinuclear staining pattern (=golgi/ER?)
4) Sample preparation:
Species: rat pancreatic beta cells
Type of sample: Fresh frozen sections, perfusion fixed frozen sections, PFA/formalin fixed paraffin embedded sections, cells in culture, other:
Sample preparation : fixation in formol
Positive control: unstimulated cells (MIX1), negative for DLK1
Negative control : cells treated with specific compound to trigger DLK1 expression (MIX2), confirmed at level of qPCR (80.000 fold increase at RNA level), ICC with 5 other rabbit anti DLK1 antibodies, including ab110636
5) Fixation step
Yes/No
If yes: Fixative agent and concentration : formaldehyde 3.7% in PBS
Fixation time: 30 minutes to 1 day
Fixation temperature: cold room 4°C
6) Antigen retrieval method: none, not required for the other tested antibodies, which all were very specific (see picture)
7) Permeabilization method:
Did you do a permeabilization step (details please) or add permeabilizing agent in any dilution buffers?
Permeabilizing agent and concentration:
TritonX100, 0.5% in PBS
8) Blocking agent (eg BSA, serum…): BSA 3%
Concentration
Blocking time: 30 to 60 minutes
Blocking temperature room temperature
9) Endogenous peroxidases blocked? No, not relevant
Endogenous biotins blocked? No, not relevant
10) Primary antibody (If more than one was used, describe in “additional notes”) :
Concentration or dilution 1/250, 1/1000
Diluent buffer: PBS
Incubation time: overnight, 4°C
11) Secondary antibody:
Species: donkey anti rabbit
Reacts against:
Concentration or dilution: 1/3000
Diluent buffer PBS
Incubation time: 30 minutes
Fluorochrome or enzyme conjugate: 488
12) Washing after primary and secondary antibodies:
Buffer: PBS+3% BSA
Number of washes : 3x 5 minutes
13) Detection method: 488 fluorescence microscope – DAPI for nuclei
14) How many times have you run this staining? 3x, in parallel with other DLK1 antibodies (ab110636, santacruz and an non commercial ab)
Do you obtain the same results every time? yes
What steps have you altered to try and optimize the use of this antibody? Not, other antibodies have been used as possitve control, we have no need to optimize this ab further.
Document attachment: see enclosed picture

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Answer

Thank you for these precisions.
We are still working on your complaint for ab21682 (anti-DLK1) and we will come back to you as soon as possible.
As requested, I have issued a free of charge replacement with the order number *******.
To check the status of the order please contact our Customer Service team and reference this number.
Please note that this free of charge replacement vial is also covered by our Abpromise guarantee. Should you still be experiencing difficulties, or if you have any further questions, please do not hesitate to let us know.

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Question

Dear,
We have a number of concerns:
1) We were recently adviced to buy the antibody purification kit (ab102784) to remove azide from the monoclonal ab110636 (see letter below), which we need to be able to use in cell culture as indicated. In the mean time we have bought this kit together with a FITC-labelingskit and two new batches of DLK1 antibodies (ab110636 and ab21682). After clean up with ab102784, it was found that ab110636 did not bind to the adviced collums, infact the antibody eluted already with the first step, the bidingbuffer. Only a minor fraction could be recovered in the washing buffers and elution buffers. An overview of our concentration measurements is provided in the attached file. This finding was also confirmed by ICC wish failed to provide a signal on a positive cell sample.
For ab21682 whitch was purified in parallel we did not notice this problem, but we only need this antibody as a negative control. It is Ab110636 which we should obtain in a purified, azide free form. What is possible?
One of the goals was also to label the purified ab110636 with de labeling kit, which now cannot be used.
2) ab21682 has been tested for ICC using 2 conditions, one negative and a second with a stimulated expression of DLK1, it was compared for its specificity to 5 other DLK1 antibodies (see attached figure). Our conclusion is that ab21682 is not specific for its target, but shows a high aspecific background staining, which is problematic since this antibody is mentioned in a few publications, and is as such the first choice to buy ffrom the website. It is however doubtfull whether this antibody can be used for ICC, and should not be sold for this purpose, since the "background" signal could be misinterpreted. I did not test it on paraffin samples for IHC
Ab110636 on the contrary is highly specific.
What could you propose?

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Answer

Thank you very much for your email and your patience.
In regards to the first issue, with the purification of ab110363, I had contacted the laboratory. There is nothing special with this clone. The only thing which might could explain the result is, that the ab110636 you received is at a low concentration. Therefore it could be too dilute to bind and elute efficiently. I can therefore propose to send you a new vial of ab110363 and a purification kit for tissue culture supernatant ab109207 for free, so you can perform the experiment. Please let me know whether you would like to accept this offer.
In regards to the ab21682 staining in ICC, I would be pleased to investigate this further. Can you please provide me with an image and the protocol details? I have attached a questionnaire which will help you to put the protocol details together. Once I have received this information, it will be easier for me to investigate and troubleshoot the results, as well as initiate internal actions if necessary. The specificity of the product is obviously very important. If the product is faulty, we will also replace it for you.
Thank you very much for your cooperation. I am looking forward to hear back from you.

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Answer

Thank you for your answer and confirming that your customer added the modifier. The modifier step is inherent to the protocol.
I have to repeat that this kit can only be used for purified antibodies. The antibodies need to be purified before the conjugation reaction.
ab102885 will label any available amine with FITC. ab91655 is not purified and therefore contains a lot of proteins that have free amines. All these free amines will interfere with the conjugation of the antibody.
I would like to summarize that ab102885 has to be purified before being labeled with the EasyLink FITC Conjugation Kit. The purification can be done with ab109207 (Antibody TCS Purification Kit):
https://www.abcam.com/index.html?datasheet=109207 https://www.abcam.com/index.html?datasheet=109207.
I hope this information is helpful for your customer.

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