The Magnetic Conjugation Kit allows one step conjugation of antibodies, proteins and peptides (or any other biomolecule with an amine group) to specially treated magnetic particles without the need for extensive optimization of the conjugation reaction; ideal for use with the 8-tube Magnetic Separation Stand.
The 0.5µm specially treated iron oxide magnetic particles offer you the right balance between high surface area and fast separation. The binding capacity of our magnetic particles is greater than other magnetic particles on the market and twice that of leading competitors. This allows you to reach the desired sensitivity with fewer particles and therefore minimizes the risk of nonspecific binding.
Covalent attachment of antibodies to magnetic particles is ideal for immunoprecipitation experiments and provides a more stable alternative to noncovalent attachment of antibodies using protein A or protein G resin.
Simply pipette your antibody into the vial of magnetic particles and incubate with constant mixing for 30 minutes. After removing the supernatant and washing, quench the reaction for 30 minutes with constant mixing. Finally wash and store the magnetic particle conjugate in storage buffer. The entire procedure requires only three minutes hands on time and your conjugate will be ready to use in a total time of one hour. The conjugation procedure has been optimized to have minimal / no unbound antibody at the end of the conjugation reaction.
Figure 1. Rabbit IgG immunoprecipitation and detection by Western blot. 25ug and 5ug Goat antirabbit antibody were added to each of two Mini vials of magnetic particles, and to 25ul of 1% magnetic beads from a leading competitor, and processed as per the respective protocols. The conjugates were used to immunoprecipitate rabbit IgG from rabbit serum. The eluted samples were run by SDS PAGE and analysed by Western blot against rabbit IgG. Expedeon’s Magnetic Conjugation Kit has a greater binding capacity and coupling efficiency than the competitor products, and allows you to detect the antigen of interest with fewer reagents.
Figure 2. Magnetic Conjugation Kit binding capacity. Goat antirabbit antibody was conjugated to 25ul of 1% magnetic particles from Expedeon’s conjugation kit (1h procedure), and to magnetic particles from a leading competitor (4h procedure) as per the respective protocols. The quantity of IgG added to the magnetic particle Expedeon’s Magnetic Conjugation Kit demonstrates a considerably greater binding capacity.
Figure 3. Rabbit IgG immunoprecipitation. Goat antiRabbit was labeled with four different batches of magnetic beads. The conjugates were tested in parallel on a Rabbit IgG immunoprecipitation. The bound Rabbit IgG was eluted and quantified by 280nm absorbance.
1.How many magnetic particles are in each kit?
A Mini vial provides 25µl 1% conjugated particles, and a Midi vial provides 250ul 1% conjugate. The amount of conjugate needed is very much dependent on the downstream application.
Yes. We have tested antibodies from a variety of species including mouse and goat.
3. Is the kit suitable for conjugating small molecules including analytes?
The Magnetic Conjugation Kit has been optimized for antibodies, but it may be possible to conjugate small molecules with primary amine functional groups. Please contact our technical support team for information and support.
4. What can I do if my antibody is not in a compatible buffer?
PBS affects the conjugation reaction to a certain extent when added neat to the Mini vial. The interference is overcome if PBS is diluted 1:2 in the reaction mixture, therefore the antibody should be at least twice as concentrated as the requirement. The conjugation reaction is compatible with most other buffers that have a pH between 7 and 9, and which have a lower concentration than 100mM. Sodium azide at a concentration up to 0.1% in the reaction mix can be tolerated. The buffer shouldn’t contain amines as these will interfere with the reaction. Should your buffer not be compatible with the Magnetic Conjugation Kit, you can use the AbPure™ antibody Concentration & Clean-up Kit to exchange the antibody buffer.
5. What can I do if my antibody has non-antibody protein components or is not purified?
To remove interfering proteins before conjugation, or to purify your antibody from ascites fluid, serum or tissue culture supernatant, please see our AbPure™ range of purification products.
6.What can I do if my antibody is 0.4mg/ml or less?
If your antibody buffer is compatible with the conjugation reaction, you can add it without further dilution into the Mini vial. We recommend adding 50µl of 0.4mg/ml concentrated antibody which corresponds to 20µg per vial, however an antibody concentration range of 0.2-1mg/ml can be explored. The volume of antibody will affect the rate of reaction, so we advise to maintain the volume at 50µl. Please contact our technical support team if your antibody is too dilute to use.
7. How can I prevent unspecific binding?
It is beneficial to add a slight excess of antibody to the magnetic particles to fully cover the surface. The quencher provided blocks any reactive groups that are left at the end of the conjugation, however BSA can be spiked with the quencher to further prevent non-specific binding.
The Mini kits are convenient for the rapid production of small quantities of conjugate for screening purposes. For applications requiring greater amounts, our Midi kit may be suitable, which contains 10x more magnetic beads than a mini vial. For bulk quantities please get in touch.
9. How do I know that the conjugation has worked?
The best way to test your conjugate is to use it in your assay. However you can confirm and estimate the amount of antibody bound to the beads by Bradford protein assay. Analyze your starting antibody mix, if free from any other protein, and the supernatant containing the unbound antibody by preparing a standard curve using a protein that has a similar weight (ideally the same protein used for the conjugation). Calculate the difference between the loaded protein and the unbound protein to estimate the coupled antibody.
Covalent conjugation of antibodies to magnetic beads in one easy step.
Request a quote now to conjugate your required antibody at your agreed conjugation ratio and scale.