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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.
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.
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
|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|
Please note: All products are "FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE"