All lanes : Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638) at 1/5000 dilution

Lane 1 : Wild-type HEK293 whole cell lysate
Lane 2 : SMARCC1 knockout HEK293 whole cell lysate
Lane 3 : HeLa whole cell lysate
Lane 4 : Jurkat whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 123 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab172638 observed at 123 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab172638 was shown to recognize in wild-type HEK293 cells as signal was lost at the expected MW in SMARCC1 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SMARCC1 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab172638 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/5000 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

Immunocytochemistry/ Immunofluorescence analysis of HeLa (Human cervix adenocarcinoma epithelial cell) cells labeling SMARCC2 with Purified ab172638 at 1:100 dilution. Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with Ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor ® 594) 1:200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor ® 488) was used as the secondary antibody at 1:1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

Flow Cytometry analysis of Jurkat (Human T cell leukemia T lymphocyte) cells labeling SMARCC1/BAF155 with purified ab172638 at 1:30 dilution (red). Cells were fixed with 4% Paraformaldehyde. A Goat anti rabbit IgG (Alexa Fluor ® 488) secondary antibody was used at 1:2000 dilution. Isotype control - Rabbit monoclonal IgG (Black). Unlabeled control - Cell without incubation with primary antibody and secondary antibody (Blue).

Chromatin was prepared from MDA-MB-231 cells according to the Abcam X-ChIP protocol. Cells were fixed with formaldehyde for 10 minutes.
The ChIP was performed with 25 µg of chromatin, 5 µg of ab172638 (red), and 20 µl of Protein A/G sepharose beads. 5 µg of rabbit normal IgG was added to the beads control (gray). The immunoprecipitated DNA was quantified by real time PCR (Taqman approach for active and inactive loci).
Primers and probes are located in the first kb of the transcribed region.

All lanes : Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638) at 1/5000 dilution (purified)

Lane 1 : HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates
Lane 2 : Rat testis lysates

Lysates/proteins at 15 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/20000 dilution

Predicted band size: 123 kDa
Observed band size: 155 kDa why is the actual band size different from the predicted?

Blocking and diluting buffer: 5% NFDM/TBST.

ab172638 (purified) at 1:30 dilution (2μg) immunoprecipitating SMARCC1/BAF155 in Jurkat whole cell lysate.

Lane 1 (input): Jurkat (Human T cell leukemia T lymphocyte) whole cell lysate, 10μg
Lane 2 (+): ab172638 & Jurkat whole cell lysate
Lane 3 (-): Rabbit monoclonal IgG (ab172730) instead of ab172638 in Jurkat whole cell lysate

For western blotting, VeriBlot for IP Detection Reagent (HRP) (ab131366) was used for detection at 1:1000 dilution.
Blocking and diluting buffer: 5% NFDM/TBST."

Western blot analysis on immunoprecipitation pellet from Jurkat cell lysate using unpurified ab172638 at a 1/10 dilution.

Flow cytometric analysis of permeabilized Jurkat cells using unpurified ab172638 at a 1/10 dilution (red) or a rabbit IgG (negative) (green).

All lanes : Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638) at 1/1000 dilution (unpurified)

Lane 1 : HeLa cell lysate
Lane 2 : K562 cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : 293T cell lysate

Lysates/proteins at 10 µg per lane.

Predicted band size: 123 kDa