Product nameAntibody TCS Purification Kit
See all Antibody Purification kits
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.
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.
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.
Note: Elute the antibody in 1ml fractions.
- 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.
- 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
Storage instructionsStore 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
ab109207 has not yet been referenced specifically in any publications.