Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade

10 product images

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

  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.

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  Western blot - 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 Anti-GAPDH antibody [6C5] - 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 Human SMARCC1 (BAF155) knockout HeLa cell line ab264859 (SMARCC1 knockout cell lysate Human SMARCC1 (BAF155) knockout HeLa 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 h at room temperature before imaging.

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

  Lane 1: Wild-type HeLa cell lysate at 20 µg

  Lane 2: SMARCC1 knockout HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human SMARCC1 (BAF155) knockout HeLa cell line (Human SMARCC1 (BAF155) knockout HeLa cell line ab264859)

  Performed under reducing conditions.

  Predicted band size: 123 kDa

  Observed band size: 112 kDa

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

  Lanes 1 - 4: Merged signal (red and green). Green - ab172638 observed at 123 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773 and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

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

  Lane 1: Wild-type HEK293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

  Lane 2: SMARCC1 knockout HEK293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 20 µg

  Lane 2: Western blot - Human SMARCC1 (BAF155) knockout HEK-293 cell line (Human SMARCC1 (BAF155) knockout HEK-293 cell line ab261854)

  Lane 3: HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 20 µg

  Lane 4: Jurkat (Human T cell leukemia cell line from peripheral blood) whole cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 123 kDa

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

  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 Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor ® 594) 1:200 (2.5 μg/ml). Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) 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.

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  Flow Cytometry (Intracellular) - Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638)

  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).
  

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

  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 (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab172638 in Jurkat whole cell lysate
  
  For western blotting, VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) was used for detection at 1:1000 dilution.
  Blocking and diluting buffer: 5% NFDM/TBST."

  All lanes: Immunoprecipitation - Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638)

  Predicted band size: 123 kDa

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

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

  All lanes: Immunoprecipitation - Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638)

  Predicted band size: 123 kDa

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

  Blocking and diluting buffer: 5% NFDM/TBST.

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

  Lane 1: HeLa (Human cervix adenocarcinoma epithelial cell) whole cell lysates at 15 µg

  Lane 2: Rat testis lysates at 15 µg

  Secondary

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

  Predicted band size: 123 kDa

  Observed band size: 155 kDa

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  Flow Cytometry (Intracellular) - Anti-SMARCC1/BAF155 antibody [EPR12395] - ChIP Grade (ab172638)

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

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

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

  Lane 1: HeLa cell lysate at 10 µg

  Lane 2: K562 cell lysate at 10 µg

  Lane 3: Jurkat cell lysate at 10 µg

  Lane 4: 293T cell lysate at 10 µg

  Predicted band size: 123 kDa