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Mouse BRM/ SMARCA2 fusion protein derived from within residues 48-214 of the corresponding human sequence.(Note: Please see Muchardt C et al. EMBO J 15:3394-402 (1996) for further details).
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|IP||Use at an assay dependent concentration.|
|ICC/IF||Use at an assay dependent concentration.|
|IHC-FoFr||Use at an assay dependent concentration. PubMed: 23359715|
|ChIP||Use at an assay dependent concentration.|
|WB||Use a concentration of 0.95 µg/ml. Detects a band of approximately 180 kDa (predicted molecular weight: 180 kDa).|
The WB image shows a composite of 2 western blots illustrating the specificity of the ab15597. The cell lines used here are as follows:
SW13- a human adrenal adenocarcinoma deficient in Brg1 and Brm (Neg.
ab15597 staining BRM (red) in Rat E17 cortical neurons 7DIV by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with formaldehyde, permeabilized with 0.5% Triton + 3% BSA and blocked with 3% BSA for 30 minutes at 20°C. Samples were incubated with primary antibody (1/100 in PBS + 3% BSA) for 1 hour at 20°C. An Alexa Fluor® 555-conjugated Donkey anti-rabbit polyclonal (1/300) was used as the secondary antibody. Dapi is stained blue and MAP2 (1/500) stained green
A) ab15597 specifically immunoprecipitates Brm. Immunoprecipitations were performed on bacculovirus–expressed Flag-tagged Brm and Brg1 from insect cells. 2µg of ab15597 (left hand side) and 2µg of J1 (right hand side) were used for each IP. J1 is a polyclonal antibody recognising both, Brg1 and Brm.
B) ab15597 chromatin immunoprecipitates Brm. Two cell lines 1 and 2 were treated with different stimuli and subjected to the ChIP procedure. Cells were fixed with formaldehyde for 10mins. The ChIP was performed with 2µg of 15597 or J1 and 10µl of protein G Dynal beads. The immunoprecipitated DNA was quanified by real-time PCR with primers specific for genes A and B, respectively. J1 is a polyclonal antibody recognising both, Brg1 and Brm.
Immunofluorescence using the Brm antibody ab15597.
It appears the antibody works well in IF detecting BRM in D98oR WT cells. The D98oR BB10 blank omitted the primary antibody as a control. However, it was also positive on a cell line that has Brm knocked down by RNAi, (the BB10 cell line). This may be because RNAi is simply a knock down not a knock out or could be that the antibody is so good it is picking up the small amount of Brm remaining in the BB10s (highly possible), or the dilution is too high. Or, it may be that its crossreacting with another protein (though there was no cross rectivity with Brg1 in WB).
Original magnification 400x. ab15597 dilution 1:100.
Work is continuing on characterising this ab in IF.