Product Information

Immunoprecipitation (IP) is an experimental technique that can purify and separate target proteins by utilizing the affinity-binding properties of antigens and antibodies. The kit contains all reagents for immunoprecipitation and subsequent Western blotting detection. It provides the best solution for immunoprecipitation experiments by efficiently and rapidly separating the concentrated target proteins. Meanwhile, the kit also has the advantages of time saving, high efficiency, and low antibody dosage and so on.

About the Kit

(1) IP lysis buffer can effectively extract total protein of cells or tissues for subsequent experiments.

(2) rProtein A/G beads slurry is used to separate antigen-antibody complexes by precipitation.

(3) Elution buffer with high salt and low pH is effectively for the dissociation of antigen-antibody complexes and rProtein A/G beads.

(4) The use of spin columns makes the operation more convenient and faster, and reduces the loss of target proteins, while reducing the influence of non-specific proteins.

(5) HRP-conjugated Protein A secondary antibody is used for the detection of Western blotting, which can effectively eliminate the influence of the light chain signal of the antibody and reduce  the heavy chain signal of the antibody.

(6) The kit can be widely used in immunoprecipitation (IP) and immunocoprecipitation (CoIP) experiments.

Components

*The customer must provide the following:

Cells or tissue for immunoprecipitation, antibodies for immunoprecipitation, 1x PBS buffer, vertical rotary mixer, low-temperature centrifuge, adjustable pipette and pipette tips, deionised water, and homogeniser (for tissue samples).

 *It is recommended not to use the kit after expiration.  

Usage

Protocol for Immunoprecipitation Kit (With HRP-conjugated protein A）

Cautions

*The kit is only used for IP and CoIP experiments of cell or tissue lysates.

*Unless otherwise specified, all operations were done on ice.

*Avoid skin and eyes contact with the Alkali neutralizing solution.

Questions and Answers

Questions 1: Target protein not obtained ?

Reason a: High concentration of detergent affects the antigen-antibody binding.

Solution a: Prepare high-concentration cell or tissue lysates and dilute with incubation buffer before use.

Reason b: Low expression or degradation of target protein in lysates.

Solution b: Increase the amount of total protein of lysates used; Verify target protein expression in the lysate by Western blotting; Replace with fresh protease inhibitors.

Reason c: The antibody does not bind to the corresponding antigen.

Solution c: Replace antibodies that recognize different epitopes and increase the amount of antibodies used.

Reason d: Antigen-antibody complexes are not precipitated by rProtein A/G beads.

Solution d: Antibodies belong to IgM subtype, and antigen-antibody complexes are precipitated using anti-IgM-conjugated microparticles.

Questions 2: Antibody signal of eluate affects the signal of target protein ?

Reason: The molecular weight of target protein is around 45-55 kDa.

Solution: Choose to use HRP-conjugated Mouse Anti-Rabbit IgG Light Chain Specific secondary antibody (when both capture and detection antibodies are rabbit antibodies); Two different species of antibodies were used for IP and subsequent Western blotting analysis respectively.

How to choose a secondary antibody for immunoprecipitation

Cited in Article as

pk10007, Immunoprecipitation Kit (With HRP-conjugated protein A), Proteintech, IL, USA