A novel tool for isolating antigen free from antibody. The kit immobilizes purified antibody onto beads to create a covalently permanent affinity support, which may be re-used up to 10 times. The technology eliminates the need for Protein A or Protein G agarose beads. An immune complex is formed when the crude sample is incubated with the beads covalently bound antibody. After several washes of the immune complex the non-bound material is removed. The elution step dissociates the bound antigen from the complex without disocciation of the antibody from the beads.
Applications
Immunopreciptations
Co-immunoprecipitations
Eliminating interference from antibody heavy and light chan bands on SDS-PAGE or Western blots
Performance Results
Gels marking
(M): Protein molecular weight marker
(B): 5% of the flow through volume from the whole HeLa cell extract after binding reaction
(Washing #1,2 and 3): 3% of washing volume number 1,3 and 5 (five washings were performed)
(Elutin #1,2 and 3): 50% from the elution volume number 1,2 and 3
(Elution with 2X PLB, X): 100% from the elution volume with 2X Protein Loading Buffer
Figure 1: Immunoprecipitation of actin from whole HeLa cell extract with IPeX kit.
(A) Immunoprecipitation of actin with monoclonal mouse anti-actin (2 g) (ICN) covalently coupled to 15 l of IPeX beads for 4 hr at room temperature and stabilized for 30 min in room temperature. 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 l) (total protein 4 g/ l), mixed with mouse anti-actin-coupled to IPeX beads (15 l of antibody-coupled IPeX beads) for 4 hr at 4oC (also, see schematic short protocol below)
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)
Products and Pricing
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TRIAL Gerard IPEX Kit includes 2 spin columns, 2 collection tubes, beads, buffers and handbook
