Immunoprecipitation Kit (With HRP-conjugated protein A)

Cat no : PK10007



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

Components20 times

Storage

IP lysis buffer : 30 ml/bottle

1 bottle

6 months at RT

Incubation buffer : 20 ml/bottle

1 bottle

6 months at RT

100 × Protease inhibitor : 2 ml /vial

1 vial

6 months at -20 ℃

20 × Washing buffer : 20 ml /bottle

1 bottle

6 months at RT

rProtein A/G beads slurry : 1 ml/vial

1 vial

6 months at 4 ℃

Elution buffer : 2.5 ml/vial

1 vial

6 months at 4 ℃

Alkali neutralization buffer : 250 μl/vial

1 vial

6 months at

5 × Sample buffer : 800 μl/vial

1 vial

6 months at RT

HRP-conjugated Protein A : 200 μl/vial

1 vial

6 months at

Spin columns : 1 ml

20 units

12months at RT

Collection tubes : 2 ml

20 units

12months at RT

End caps

20 units

12months at RT

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

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

Capture Antibody Types for IP

Primary Antibody Types for WB

Secondary Antibody Types for WB

Mouse McAb/PcAb

Mouse McAb

HRP-conjugated Protein A or HRP-conjugated anti-Mouse IgG secondary antibody

Mouse PcAb

Rabbit antibody

HRP-conjugated anti-Rabbit IgG secondary antibody

Rabbit antibody

Mouse McAb

HRP-conjugated anti-Mouse IgG secondary antibody

Mouse PcAb

Rabbit antibody

HRP-conjugated Protein A; HRP-conjugated anti-rabbit IgG light chain specific antibody

Cited in Article as

PK10007, Immunoprecipitation Kit (With HRP-conjugated protein A), Proteintech, IL, USA

Publications

ApplicationTitle

Redox Biol

p38α in macrophages aggravates arterial endothelium injury by releasing IL-6 through phosphorylating megakaryocytic leukemia 1.

Authors - Meng Zhang

Cell Biol Toxicol

AS3MT facilitates NLRP3 inflammasome activation by m6A modification during arsenic-induced hepatic insulin resistance.

Authors - Tianming Qiu

Ecotoxicol Environ Saf

NLRP3 inflammasome blocked the glycolytic pathway via targeting to PKLR in arsenic-induced hepatic insulin resistance.

Authors - Jingyuan Zhang
CoIP

Cancer Cell Int

Leptin Receptor (LEPR) promotes proliferation, migration, and invasion and inhibits apoptosis in hepatocellular carcinoma by regulating ANXA7.

Authors - He Huang

Toxicology

Resveratrol protected acrolein-induced ferroptosis and insulin secretion dysfunction via ER-stress- related PERK pathway in MIN6 cells.

Authors - Xinhao Zhang

Cell Signal

IL-6/STAT3 signaling activation exacerbates high fructose-induced podocyte hypertrophy by ketohexokinase-A-mediated tristetraprolin down-regulation.

Authors - Jie Zhou