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Recombinant fragment derived from residues 100 - 200 of Human EMSY.
Our Abpromise guarantee covers the use of ab123 in the following tested applications.
The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.
|WB||1/3000. Detects a band of approximately 141 kDa (predicted molecular weight: 141 kDa). Variability in the size at which EMSY runs by Western blot has been experienced with different protein marker systems. EMSY has been observed to run at between 115 and 150kD. However, the independently raised ab4579 has been used to confirm the specificity of this antibody.|
|IP||Use a concentration of 5 µg/ml.|
|IHC-P||1/500. Perform heat mediated antigen retrieval via the pressure cooker method before commencing with IHC staining protocol.|
EMSY was immunoprecipitated using 0.5mg MCF7 whole cell extract, 5µg of Rabbit polyclonal to EMSY and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, MCF7 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70°C; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab123.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 141kDa; EMSY
Hughes-Davies et al, 2003
WB using EMSY antibody (ab123) demonstrating the interaction between EMSY and BRCA2 by immunoprecipitation.
Endogenous coimmunoprecipitation of EMSY and BRCA2 from unmanipulated asynchronously dividing HeLa cells.
Immunoprecipitation with an unrelated antibody (anti-GFP), preimmune serum, ab123 or anti-BRCA2 antibody was followed by Western blotting with ab123.
Neither of the unrelated antibodies could coprecipitate EMSY, whereas an EMSY signal was detected in the immunoprecipitate obtained with the anti-BRCA2 antibody. The anti-EMSY antibody could also immunoprecipitate endogenous EMSY protein.
Upper panel: EMSY responds to DNA damage.
EMSY antibody (ab123) immunofluorescence staining of immortalized mouse wild-type embryonic fibroblasts before and after treatment with ionizing radiation (10 Gray at 1.8 Gy/minute, 250 kVp). EMSY assembles into nuclear speckles over several hours.
Lower panel: Costaining with a monoclonal mouse antibody to gamma-H2AX reveals that EMSY relocalizes to DNA damage sites.
Western blot of EMSY on MCF-7 cell lysate.
Lane 1: ab4579 at 1/500
Lane 2: ab123 at 1/500.
Variability in the size at which EMSY runs by Western blot has been experienced with different protein marker systems. EMSY has been observed to run at between 115 and 150kD (even though the predicted size is 141kD). However, that the same size band is detected by both ab123 and ab4579 (raised with immunogens of different sequence) confirms the specificity of these antibodies.
Secondary ab Lane 1: Rabbit polyclonal to Goat IgG H&L (HRP) ab6741 (1/5000)
Exposure time: 3 min.
Secondary ab Lane 2: Goat polyclonal to Rabbit IgG H&L (HRP) Pre-Adsorbed ab7090 (1/5000)
Exposure time: 10 sec.
Lysate at 20
Lane 1 - Exposure time : 3 min.
Lane 2 - Exposure time : 10 sec.
Variability in the size at which EMSY runs by Western blot has been exper