Immunoprecipitation of Actin Free from Antibody

 

 

 

 

 

 

 

 

 

 

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Overview

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

Product Description

Product Number
# of preps

Price
(Click to Order)

TRIAL Gerard IPEX Kit includes 2 spin columns, 2 collection tubes, beads, buffers and handbook

IP-2

2

$24

Gerard IPEX Kit includes 2 spin columns, 2 collection tubes, beads, buffers and handbook

IP-10

10

$120

Refill Kit for 100 preparations
IPR-100
100

 

Protocol

 

 
 
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