7. microRNA Research

for Immunoprecipitation, Western Blot, Immunocytochemistry
Anti Human Ago2, Monoclonal Antibody(Clone No. 4G8)
Wako Cat. #011-22033 (50µL); 015-22031 (100µL)
Keep at 2~10℃


Argonaute2(Ago2) was isolated as one of the main components of RISC (RNA-induced silencing complex). Ago2 captures siRNA and microRNA which are working as a guide molecule for interaction with target mRNAs in RNAi pathway. In this pathway, Ago2 catalyzes the nicking of target mRNAs and binding between RISC and target mRNAs. This monoclonal antibody is not only used for western blot and immunocytochemistry (ICC), but also immunoprecipitation (IP) of hAgo2.
ApplicationWorking Dilution
Western Blot1 : 100 - 1 : 200
Immunoprecipitation1:50
Immunocytochemistry1 : 20 - 1 : 50

OrganismsHumanMouseHamsterRat
CellHeLaP388D1CHOSCC-131
Western BlotOXXX
ImmunoprecipitationOXXX
ImmunocytochemistryOXXX
microRNA Purifi cationOXXX
[Features]
1. For IP, ICC, Western Blot
2. Specifi c reactivity with human Ago2 protein
3. For purifi cation of RNA captured by RISC
Concentration (protein): Indicated on the label.
Formulation: 0.09% Sodium Azide, 10% Glycerol
with 1 x TBS, pH7.4.
Subclass: IgG1
Antigen: Recombinant human Ago2
Storage: 2~10℃ in the dark. Avoid the freeze and thaw.
[References]
1) Qi, H. H., et al.: Nature, 455 (7211), 421 (2008)
2) Miyoshi, K., et al.: Methods Mol. Biol., 442, 29 (2008)
3) Azuma-Mukai A., et al.: Proc. Natl. Acad. Sci. U. S. A., 105(23), 7964 (2008)
APPLICATION DATA
Immunoprecipitation of hAgo2 protein from HeLa cell line
image
[ Figure 1 ]
(A) Western blot of hAgo2 protein from HeLa cell lysate. The band of hAgo2 protein was detected in approximately 100kDa. Working dilution of this product was 1/100 dilution. Cell number was 5-10 x 107 cells.
(B) Immunoprecipitation of hAgo2 protein from HeLa cell lysate by using Gamma-bind beads immobilized with this antibody. The band of hAgo2 protein was detected in approximately 100kDa by using silver staining. Cell number was 5 x 106 cells.
Immunocytochemistry of hAgo2 protein of HeLa cell line
image
[ Figure 2 ]
Immunocytochemistry of hAgo2 protein of HeLa cell line by using 1/50 diluted this antibody. hAgo2 protein was localized in P-body, indicated by arrows, and cytoplasm.

Specifi city of anti hAgo2, Monoclonal Antibody (4G8)
image
[ Figure 3 ]
Immunoprecipitation of hAgo2 protein from human cultured cell lines (HeLa, HepG2, HEK293, THP-1) and mouse cultured cell line (P388D1) by using 20µL 10% Protein G slurry immobilized with 10µg this antibody. The bands of hAgo2 protein was detected in approximately 100kDa by using silver staining and western blot. Cell number was 5×106 cells.
<Lane M: MW marker; Lane 1: HeLa (Human); Lane 2: HepG2 (Human);
Lane 3: HEK 293 (Human); Lane 4: THP-1 (Human); Lane 5: P388D1 (Mouse)>


image
[ Figure 4 ]
Purifi cation of microRNA fraction from immunoprecipitated hAgo2 protein. The purifi ed microRNA fraction from human cultured cell lines (HeLa, HepG2, HEK293, THP-1) were specifically detected by Urea-PAGE. Cell number of each cell line is 5×106 cells. The applied volume per lane is half of 10µL of fi nal solution prepared with an IP.
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