Advantages in using an IPeX kit rather than classical IP methods:

• The affinity support can be reused up to 10 times.
• Immobilizing the antibody provides faster and easier IP's.
• Purified antigen free from antibody contamination.
• Coupling of all primary amine-containing molecules.
• Coupling of all antibody species and subclasses.
• Antibody is coupled directly to the beads without cross-linker.
• Detection of assay proteins close to or of similar size as the heavy and light chains using Western blots.
  

PRODUCT CATALOG REFERENCE

Products Description	KIT Contents	Cat. No.
IPeX kit (for 10 preparations)	10 IPeX spin columns, 10 collection tubes, beads, buffers and handbook	IP-10
Gentle Elution Buffer	2 ml of Gentle Elution Buffer	GEB-2
Gentle Elution Buffer	20 ml of Gentle Elution Buffer	GEB-20
IPeX refill kit (for 100 preparations)	20 ml of Binding buffer, 350 ml of Washing buffer and 20 ml Elution Buffer	IPR-100

IPeX Immunoprecipitation Kit, for protein purification, immobiliz proteins directly onto beads, creating a covalently permanent affinity support. IPeX  technology eliminates Protein A or Protein G agarose beads. Immune complex is formed when crude samples is incubated with the beads covalently bound antibody. 

Elution step dissociated the bound antigen from the complex  without dissociation of  antibody from beads. Once beads are pre activated, beads can be re used up to 10 times.

Immunoprecipitation of actin from whole HeLa cell extract with IPeX kit: (A) Immunoprecipitation of actin with monoclonal mouse anti-actin (2 g) covalently coupled to 15 µl of IPeX beads for 4 hr at room temperature (RT) and stabilized for 30 min in RT. 100% of the antibody was coupled (for detailed protocol see Gene Bio-Apllication Ltd. IPeX handbook) (also, see schematic short protocol). (B) Immunoprecipitation of actin using IPeX beads was preformed identically without the antibody as a control for antibody specific binding.

The immunoprecipitation of actin was preformed with a whole HeLa cell extract (100 µl) (total protein 4 g/µl), mixed with mouse anti-actin-coupled to IPeX beads (15 µl of antibody-coupled IPeX beads) for 4 hr at 4^oC (for detailed protocol see IPeX handboo). Immunoprecipitation and coupling of the antibody to the IPeX beads were monitoring by sampling different steps of the reactions (in this experiment: Washing and Elution monitoring was preformed), separating the samples on 12% SDS-PAGE, transfer the protein(s) to nitrocellulose membrane by a semi dry blotter. Western blot of actin is performing by using monoclonal mouse anti-actin as first antibody (1:5000 dilution) and mouse anti-IgG conjugated to HRP as second antibody (1:6000 dilution).

Gels marking

(M): Protein molecular weight marker.
(B): 5% of the flow through from the whole HeLa cell extract after biding reaction.
(Washing # 1,2 and 3): 3% of washing number 1,3 and 5 (five washing were preformed).
(Elution # 1,2 and 3): 50 % from the elution volume number 1,2 and 3.
(-): Space in loading of the gel.
(Elution with 2X PLB, X): 100% from the elution volume with 2X Protein Loading Buffer.