Name: Co-Immunoprecipitation (Co-IP) Kit
SKU: BCIPKT
Price: 450.00 USD
Availability: InStock

Co-Immunoprecipitation (Co-IP) Kit

$450.00

Item Cat No.: BCIPKT

Application: Co-Immunoprecipitation

Reactivity: Cell lysate

BiCell Scientific’s Co-Immunoprecipitation (Co-IP) Kit enables isolation of target proteins from a lysate or other complex mixtures by covalently crosslinking antibodies onto Protein A/G/L resin.

1 kit (50 reactions)

Co-IP is an important approach to study protein:protein interactions that uses an antibody to immunoprecipitate the antigen (bait protein) and co-immunoprecipitate any interacting proteins (prey proteins). Traditional co-IP methods that use Protein A or G resin result in co-elution of the antibody heavy and light chains that migrate with relevant bands on PAGE gel, masking important results.

BiCell Scientific’s Co-IP Kit resolves this issue by covalently crosslinking antibodies onto protein A/G/L resin with DSS (disuccinimidyl suberate). The addition of protein L resin into the kit improves its binding efficacy to rat antibodies. The kit also includes optimized buffers for protein solubilizing, binding and recovery.

The kit contains:

1 ml of Protein A/G/L resin (supplied as a 50% slurry)
50 ml of Resin Coupling Buffer (10 mM sodium phosphate, 150 mM NaCl, pH 7.5)
250 μl of Crosslinking Buffer (10 mM DSS)
10 ml of Cell Lysis Buffer (50 mM Tris, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1% NP-40, pH 7.5)
50 ml of Co-IP Wash Buffer (50 mM Tris, 500 mM NaCl, 1 mM EDTA, 0.5% Triton X-100, 0.5% NP-40, pH 7.5)
1 ml of SDS Loading Buffer (50 mM Tris, 2% SDS, 100 mM DTT, 1 mM Bromophenol blue, pH 6.5)

Store at 4^oC

For Research Use Only. Not for use in clinical diagnostics.

Antibody Immobilization

(The following protocol is optimized for crosslinking 10 μg of antibody to 20 μl resin.)

Gently swirl the bottle of Protein A/G/L resin to obtain an even suspension. Aspirate 20 μL of the resin slurry into a new Eppendorf tube.
Centrifuge resin at 3,000 xg for 1min. Wash the resin with 200 μl of Resin Coupling Buffer and centrifuge again.
Resuspend the resin pellet with 100 μl of Resin Coupling Buffer. Add 10 μg of antibody to the resin solution.
Incubate the antibody-resin solution on a rotator at room temperature for 30 minutes, ensuring that the slurry remains suspended during incubation.
Centrifuge resin at 3,000 xg for 1min. Wash the resin with 200 μl of Resin Coupling Buffer and centrifuge again. Repeat this step two more times.
Resuspend the antibody-resin pellet with 100 μl of Resin Coupling Buffer. Add 5 μl of Crosslinking Buffer.
Incubate the antibody-resin solution on a rotator at room temperature for 30 minutes, ensuring that the slurry remains suspended during incubation.
Centrifuge resin at 3,000 xg for 1min. Wash the resin with 200 μl of Resin Coupling Buffer and centrifuge again. Repeat this step two more times.
Chill resin pellet on ice or proceed to Co-IP reaction.

Cell Lysis

Centrifuge cell suspension at 1000 ×g for 5 minutes to pellet the cells. Discard the supernatant.
Wash cells once by suspending the cell pellet in PBS. Centrifuge at 1000 ×g for 5 minutes to pellet cells.
Add ice-cold Cell Lysis Buffer to the cell pellet. Use 500 μL of Cell Lysis Buffer per 50 mg of cell pellet (i.e., 10:1 v/w).
Incubate cell lysate on ice for 5 minutes with periodic pipetting. Remove cell debris by centrifugation at 10,000 ×g for 5 minutes. Transfer supernatant to a new tube for protein concentration determination.

Removing Nonspecific Binding

For 500 μl of cell lysate, add 50 μl of unbound protein A/G/L resin.
Incubate the lysate-resin solution on a rotator at room temperature for 30 minutes, ensuring that the slurry remains suspended during incubation.
Centrifuge resin at 3,000 xg for 1min.
Discard the resin and chill the supernatant on ice, which will be added to the immobilized antibody for co-IP.

Co-IP

Aliquot the pre-cleared cell lysate into 100 μl volume. Each volume will be used for one antibody pulldown. Negative control antibody is an antibody that pulls down an irrelevant protein not participating the bait-prey interaction.
Add the 100 μl volume of pre-cleared cell lysate to the pre-chilled antibody-resin pellet.
Incubate the lysate-resin solution on a rotator at room temperature for 30 minutes, ensuring that the slurry remains suspended during incubation.
Centrifuge resin at 3,000 xg for 1min. Wash the resin with 200 μl of Co-IP Wash Buffer and centrifuge again. Repeat this step two more times.
Resuspend the antibody-resin pellet with 100 μl of SDS Loading Buffer.
Heat at 95^oC for 5min. Chill on ice. Centrifuge resin at 3,000 xg for 1min.
Collect the supernatant, which will be ready for western blot.

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