Overview

  • Product name
    Mouse TCS Purification System
    See all Antibody Purification kits
  • Product overview

    Efficient mouse antibody purification from tissue culture supernatants. Processes 10-25 mL of supernatant to purify up to 1.5 mg of antibody per run.


    The mouse resin has a very high affinity and specificity for mouse IgG molecules. ab128749 Mouse TCS Purification System can be used to purify mouse IgG fractions from hybridoma supernatants. The binding strength of bovine IgG to Abcam's Mouse resin is negligible; therefore, it can be used to selectively recover mouse IgG from TCS samples.


     


    The method involves capture of the mouse antibody on the Mouse resin and removal of unwanted substances using a simple wash procedure. The purified product is eluted and neutralized.


     


    The mouse antibody to be purified should be in 10 to 25ml of tissue culture supernatant. Up to 1.5mg of antibody can be purified in each run.


     


    The components of ab128749 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 mouse antibody purification kit (ab204910).


     


    Ab128749 is suitable for use with Mouse antibodies. It is not suitable for use with antibodies from other species.

  • Notes

    Protocol

    Step 1: Prepare the tissue culture supernatant

    Step 2: Transfer the Mouse resin to the prepared supernatant and mix for 2 hours

    Step 3: Transfer the solution into the column

    Step 4: Wash the Mouse resin

    Step 5: Elute and neutralize purified antibody

    Step 6: Confirm elution using a protein assay

    Step 7: Concentrate the mouse antibody (optional)

     

    1. Preparing the tissue culture supernatant

    Add the 10x Binding Buffer to the tissue culture supernatant. The volume required is 1/10 of the volume of tissue culture supernatant. For example, to 20ml of tissue culture supernatant add 2ml of 10x Binding Buffer and mix by inversion.

    2. Incubation of Sample with Resin

    Add the Mouse resin to the supernatant and incubate with mixing at room temperature for a minimum of 2 hours. Use the supernatant to rinse the bottle to recover all of the Mouse resin.

    Note: The mouse antibody to be purified should be in 10 to 25ml of tissue culture supernatant. Up to 1.5mg of antibody can be purified in each run.

    3. Packing of the column

    Carefully pour the supernatant-resin mix into the column. Sample volumes of more than 10ml will have to be added in aliquots. The Mouse resin will collect in the bottom of the column. The 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 Wash Buffer to remove any non-bound protein. Repeat the wash procedure three times.

    Note: Wash the inner surface of the column to remove any residual starting material.

    5. Elution

    The mouse antibody is eluted in 1ml fractions. Place a collecting tube under the column and add 1ml of Elution Buffer. Remove the collection tube and add 0.25ml of Neutralization Buffer and mix. Repeat the elution process with a fresh collection tube three more times, each time neutralizing the sample as it is eluted. The Neutralization Buffer must be added as soon as possible to avoid prolonged exposure to low pH which can result in denaturation of the mouse IgG. The protein normally elutes in tubes 1 and 2 but you should confirm this using a test for protein (see below) before pooling any of the tubes.

    6. Antibody concentration (optional)

    If the concentration of the recovered mouse antibody is low then it can quickly and easily be concentrated using the antibody concentrator.

    Step 1: Add the mouse antibody to the top of the spin cartridge.

    Step 2: Spin for 1 to 3 minutes* in a microfuge at 13,000rpm (equivalent to 12,000g) to reduce the buffer volume in the spin cartridge to 50-100µl.

    Step 3: Repeat steps 1 to 2 as many times as is necessary to process the entire mouse antibody to the desired concentration. It may be necessary to discard any excess buffer collected in the collection tube between spins.

    Step 4: Recover the concentrated antibody from the top of the spin cartridge.

     

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

    *Spin times will vary depending on buffer composition and volume as well as centrifuge speed.

     

    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 c

Properties

  • Storage instructions
    Store at +4°C. Please refer to protocols.
  • Components 1 tests 3 tests
    10x Binding Buffer 1 unit 1 unit
    Concentration Spin Column and Collection Tube 1 unit 3 units
    Elution Buffer 1 unit 1 unit
    Mouse Resin 1 unit 3 units
    Neutralizer Buffer 1 unit 1 unit
    Purification Column 1 unit 3 units
    Wash Buffer 1 unit 1 unit
  • Research areas

Images

  • A representative bar graph comparing the effectiveness of Mouse TCS Purification System and Protein G resin.
  • Gel electrophoresis of TCS samples purified with the Mouse TCS Purification System.
    M = Mouse IgG1 in a TCS sample
    B = Bovine serum IgG in a TCS sample
    TCS = Tissue culture supernatant sample

Protocols

References

This product has been referenced in:
  • Di Marco Barros R  et al. Epithelia Use Butyrophilin-like Molecules to Shape Organ-Specific ?d T Cell Compartments. Cell 167:203-218.e17 (2016). Read more (PubMed: 27641500) »
  • Crawford JR  et al. Plasma Levels of Endothelial Microparticles Bearing Monomeric C-reactive Protein are Increased in Peripheral Artery Disease. J Cardiovasc Transl Res 9:184-93 (2016). Read more (PubMed: 26891844) »
See all 2 Publications for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Answer

Thank you for your reply.

I have again talked to the lab, and they have said that 6ml of 10X Binding Buffer, there should be more than enough binding buffer for your experiments and so you do not need to worry about not having enough. As for pH, as long as it within normal ranges that allow cells to grow well, you should have no issues.

If there is anything else I can help you with, please let me know.

Read More

Answer

Although the formulation of the elution buffer is considered proprietary, the laboratory offers the following information:

The elution buffer does not contain any amines or thiols. It should be noted that the customer should follow the protocol and proceed to the neutralization step after the elution step. This is because the elution buffer is of low (acidic) pH. Following neutralization there should be no amines or thiols present in the eluate.

Read More

Answer

Thank you for contacting Abcam.

I have talked to the lab and they have told me that if the amount (mg) of antibody is indeed below the capacity stated, then this product can be used with larger sample volumes (within reason).

Please let me know if there is anything else I can help you with.

Read More

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