Recombinant Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638)

Lanes 1 - 2: Merged signal (red and green). Green - ab172638 observed at 112 kDa. Red - loading control ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab172638 was shown to react with SMARCC1 in wild-type HeLa cells in Western blot with loss of signal observed in SMARCC1 knockout cell line ab264859 (SMARCC1 knockout cell lysate ab258198). Wild-type HeLa and SMARCC1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab172638 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4 °C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 h at room temperature before imaging.

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.

Intracellular 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). 

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.

Intracellular flow cytometric analysis of permeabilized Jurkat cells usingunpurified ab172638 at a 1/10 dilution (red) or a rabbit IgG (negative) (green).