Immunoprecipitation

What is immunoprecipitation?

Immunoprecipitation (IP) is a method of isolating a specific protein from a complex mixture such as a cell lysate, tissue homogenate or blood sample. The protein is captured by an antibody, and the antibody-antigen complex is pulled out of the sample by virtue of antibody attachment to a bead. When performing an IP reaction, one approach is to incubate the antibody with the beads, then to add the antibody-bead complex to the sample. An alternative method is to incubate the antibody with the antigen, then to add the beads to the mix. Once the bead-antibody-antigen complex has been formed, wash steps are used to remove any free antibody or free antigen. The washes are performed by pelleting the beads at the bottom of the tube through centrifugation, followed by careful removal of the supernatant by pipetting. At the end of the IP reaction the complex is typically heated in an SDS-PAGE sample loading buffer, then the sample is analyzed by Western blotting.

 

Immunoprecipitation process

The immunoprecipitation process. During a pre-immobilized antibody approach (upper) the antibody is incubated with the beads, and then this complex is added to the sample. A free antibody method (lower) requires incubation of the antibody with the sample, followed by addition of the beads. Wash steps are used to remove any free antibody or free antigen. The eluates are typically analyzed by Western blotting.

Protein A/G agarose beads for immunoprecipitation

The beads which are used to pull down the antibody-antigen complex are typically composed of agarose which has been coupled to either Protein A or Protein G. These proteins contain binding sites for the Fc region of mammalian IgG antibodies, however they vary in their ability to bind antibodies from different host species and sub-types. The binding specificity should therefore be taken into consideration when selecting an appropriate bead for an IP reaction.

Species

Subtype

Protein A Affinity

Protein G Affinity

Rabbit

IgG

High

High

Human

IgG

High

High

 Mouse

IgG1

Low

High

Mouse

IgG2a

High

High

 Mouse

IgG2b

High

Medium

 Mouse

IgG3

Medium

Medium

Goat

IgG

Low

Medium

Sheep

IgG

Low

Medium

Rat

IgG1

None

Low

Rat

IgG2a

None

Medium

 Rat

IgG2b

None

Medium

 Rat

IgG2c

Low

Medium

Relative affinities for Protein A and Protein G of the most commonly used antibody subtypes.

Innova Biosciences offers Protein A agarose and Protein G agarose which may be used to pull down antibody-antigen complexes in IP reactions. These are supplied as a pack size of 2ml resin, with >4mg Protein A/G per ml of resin. Should you wish to order a custom pack size, please contact us.

 

Magnetic beads for immunoprecipitation

A common problem associated with the use of Protein A/G agarose for immunoprecipitation is co-elution of the antibody with the antigen. The reason for this is that the bond which is formed between the Fc region of the antibody and the Protein A/G is non-covalent, allowing the antibody to detach from the agarose beads during elution. The antibody heavy and light chains, of approximately 55kDa and 25kDa respectively, can subsequently interfere with downstream analysis.

To overcome this issue, we have developed our Magnetic Bead Conjugation Kit. This allows one-step covalent conjugation of antibodies (or any other biomolecule with an amine group) to specially treated magnetic particles without the need for extensive optimization of the conjugation reaction.

 

The magnetic bead conjugation procedure.

Our Magnetic Bead Conjugation Kit offers several advantages:

✓ Quick and easy to use
✓ Covalent bond – highly stable antibody-bead conjugates
✓ High binding capacity – achieve high sensitivity with less material / conjugate
✓ High coupling efficiency – save precious antibody
✓ Fast and gentle washing procedure – no aggregation or antibody damage

Covalent attachment of antibodies to magnetic particles is ideal for IP experiments and provides a more stable alternative to non-covalent attachment of antibodies using Protein A/G agarose. The binding capacity of our magnetic particles is higher than that of other magnetic particles on the market, and twice that of leading competitors. Why not contact us to find out more?

Co-immunoprecipitation

Immunoprecipitation of a protein complex from a sample is known as co-immunoprecipitation, or co-IP. It relies on the use of a specific antibody to capture a protein which is known, or believed to be part of, a larger complex of proteins. Following elution of the protein complex from the antibody, the various components of the complex can be evaluated.

 

The co-immunoprecipitation process.

Lightning-Link® for Western blotting

Although Western blotting has traditionally relied on the use of labeled secondary antibodies for detection, the process can be greatly simplified by directly conjugating the primary antibodies to the relevant detection moieties. This offers several advantages:

✓ Non-specific binding is avoided since secondary antibodies are not used
✓ Multiplexing is possible with antibodies from the same species
✓ Faster since there is no secondary antibody incubation step and therefore fewer wash steps
✓ Data quality is improved through assay simplification

Lightning-Link® from Innova Biosciences is an innovative technology that enables direct labeling of antibodies (or any other biomolecule with free amine groups), with just 30 seconds hands-on time. The Lightning-Link® kits are quick and easy to use, and with no separation steps involved, 100% of the antibody is recovered at the end of the labeling process.

The use of directly labeled primary antibodies can be especially beneficial for Western blot evaluation of co-IP experiments, since the same sample on the same blot can be probed simultaneously for multiple proteins. This allows more accurate quantification of the different components of the protein complex.

Streptavidin Resin

Amintra Streptavidin Resin can be used to bind biotinylated biomolecules in a variety of batch- or column-type affinity procedures with low non-specific binding. Using an innovative immobilization technology, Amintra Streptavidin Resin offers an ultra-high biotin-binding capacity with a very low leaching.

Visit our website to find out more about Expedeon products that can assist you in Immunoprecipitation application.

 

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