-
Immunoprecipitations.
-
Co-immunoprecipitations.
- Small-scale
purifications of protein.
- Small-scale
purifications of recombinant protein (e.g.
with His or GST tags)
- Studying of
protein-protein interaction.
- Eliminating
interference from antibody heavy and light
chain bands on SDS-PAGE or Western blots.
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.
-
Detecting at
Western blots assay proteins close or of
similar size as the heavy and light
chains.
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 4oC
(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.
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