Anti-SMARCA2 / BRM antibody - ChIP Grade (ab15597)

  ChIP - BRM antibody (ab15597)

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.

Dr. Matthias Kaeser, RBIO-E/Emerson Lab, La Jolla

  Western blot - Anti-SMARCA2 / BRM antibody - ChIP Grade (ab15597)

Predicted band size : 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.Control)

D98oR Par. – a subclone of Hela (human ovarian carcinoma) that expresses both Brm and Brg1 endogenously.

D98oR HTP V4 - D98oR parental cell line stably transfected with pHTPsiRNA (vector only).

D98oR HTP Brg1i-11 - D98oR parental cell line stably transfected with pHTPBrg1i expressing an artificial gene generating a short hairpin RNAi (shRNA) targeting Brg1.

D98oR HTP Brmi-2 - D98oR parental cell line stably transfected with pHTPBrmi expressing an artificial gene generating a short hairpin RNAi (shRNA) targeting Brm.

D98oR HTP Brmi-7 – A second stably transfected D98oR parental cell line targeting the Brm with the pHTPBrmi construct.

D98oR HTP BB10 – a D98oR HTP Brg1i-11 cell line that has also been stably transfected with pHTPBrmi creating a double knockdown of Brg1 and Brm.

All cells were grown in RPMI 1640 plus 10% FBS. Whole cell protein lysates were generated using an 8M urea extraction protocol (8M urea, 0.1 M NaH2PO4, 10mM Tris, pH 8, and 14.3 mM BME) to insure maximum isolation of the subject proteins (Reyes et al, 1997). The protein concentration of the extracts was determined by the method of Bradford using the BioRad Protein Assay (BioRad Laboratories, Hercules, CA) per the manufacturer’s instructions using bovine serum albumin (BSA) as a standard. All extractions were performed on ice and lysates held at - 80 degrees C until used.

Thirty micrograms of protein lysates from each sample were size fractionated in a 7.5% precast polyacrilamide gel (PAGEr Gold, BMA, Rockland, ME) at a constant voltage of 115V. The protein was subsequently transferred to an Immobilon-P membrane (Millipore Corp., Bedford, MA) overnight at a constant current of 80mA. Separate but identically loaded blots were probed overnight at 4 degrees C with Abcam rabbit anti Brm (1:4000 dilution), a monoclonal Brg1 antibody from Santa Cruz Biotechnology, (Santa Cruz, CA; 1:1000 dilution), or a rabbit polyclonal for actin from Sigma (St. Louis, MO; 1:1000 dilution). Secondary antibodies, anti-mouse IgG and anti-rabbit IgG conjugated to horseradish peroxidase, (Amersham Life Science, Inc., Arlington Heights, IL) were used at a dilution of 1:4000. Western blots were developed using the ECL Western Blotting Detection Reagents (Amersham-Pharmacia Biotech) and exposed using Kodak ML film (Eastman Kodak Co., Rochester, NY). Brm and Brg1 exposures are approximately 1 minute.

Gary Rosson

  Immunocytochemistry/ Immunofluorescence - SMARCA2 / BRM antibody - ChIP Grade (ab15597)

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.

Garry Rosson, North Carolina at Chapel Hill

  Western blot - BRM/ SMARCA2 antibody - ChIP Grade (ab15597)

All lanes : Anti-SMARCA2 / BRM antibody - ChIP Grade (ab15597) at 1 µg/ml

Lane 1 : D98oR (subclone HeLa) positive control lysate
Lane 2 : SW13 (Human adrenal adenocarcinoma) negative control lysate

Lysates/proteins at 20 µg per lane.


Predicted band size : 180 kDa
Observed band size : 180 kDa

  Western blot - SMARCA2 / BRM antibody - ChIP Grade (ab15597)

All lanes : Anti-SMARCA2 / BRM antibody - ChIP Grade (ab15597) at 1 µg/ml

Lane 1 : Brain (Mouse) Tissue Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 3 : HEK293 (Human embryonic kidney cell line) Whole Cell Lysate

Lysates/proteins at 20 µg per lane.

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution
developed using the ECL technique

Performed under reducing conditions.

Predicted band size : 180 kDa
Observed band size : 220 kDa (why is the actual band size different from the predicted?)
Additional bands at : 35 kDa,60 kDa. We are unsure as to the identity of these extra bands.

Exposure time : 5 minutes