Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade

19 product images

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  ChIP - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Chromatin was prepared from HCT 116 cells according to the Abcam X-ChIP protocol. Cells were fixed with 1% formaldehyde for 10 minutes. The ChIP was performed with 25μg of chromatin, 5μg of ab129195 (red), and 20μl of protein A/G sepharose beads slurry (10μl of sepharose A beads + 10μl of sepharose G beads). 5μg of rabbit normal IgG was added to the beads control (grey). The immunoprecipitated DNA was quantified by real time PCR (Sybr green approach).

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  Immunoprecipitation - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  ab129195 (purified) at 1/20 immunoprecipitating KDM1/LSD1 in 10 μg Jurkat cell lysate (Lanes 1 and 2, observed at 110 kDa). Lane 3 - Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730).
  

  For western blotting, VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/10,000 dilution.

  Blocking/Dilution buffer and concentration: 5% NFDM/TBST.

  All lanes: Immunoprecipitation - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Predicted band size: 92 kDa

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  Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Lanes 1- 2: Merged signal (red and green). Green - ab129195 observed at 110 kDa. Red - Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) observed at 37 kDa.
  

  ab129195 was shown to react with KDM1/LSD1 in wild-type HeLa cells in western blot. Loss of signal was observed when knockout cell line Human KDM1A (LSD1) knockout HeLa cell line ab265790 (knockout cell lysate Human KDM1A (LSD1) knockout HeLa cell lysate ab256965) was used. Wild-type HeLa and KDM1A knockout HeLa cell lysates were subjected to SDS-PAGE. Membrane was blocked for 1 hour at room temperature in 0.1% TBST with 3% non-fat dried milk. ab129195 and Anti-GAPDH antibody [6C5] - Loading Control (Anti-GAPDH antibody [6C5] - Loading Control ab8245) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^®800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^®680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195) at 1/1000 dilution

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

  Lane 2: KDM1A knockout HeLa cell lysate at 20 µg

  Lane 2: Western blot - Human KDM1A (LSD1) knockout HeLa cell line (Human KDM1A (LSD1) knockout HeLa cell line ab265790)

  Performed under reducing conditions.

  Predicted band size: 155 kDa, 92 kDa

  Observed band size: 110 kDa, 160 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Immunohistochemical staining of paraffin embedded rat kidney with purified ab129195 at a working dilution of 1/50. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.
  

  PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

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  Immunocytochemistry/ Immunofluorescence - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  ab129195 staining KDM1A/LSD1 in wild-type HAP1 cells (top panel) and KDM1A knockout HAP1 cells (bottom panel). The cells were fixed with 4% formaldehyde (10min), permeabilized with 0.1% Triton X-100 for 5 minutes and then blocked with 1% BSA/10% normal goat serum/0.3M glycine in 0.1% PBS-Tween for 1h. The cells were then incubated with ab129195 at 1μg/ml concentration and Alexa Fluor® 594 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker ab195889 at 1/250 dilution (shown in pseudo colour red) overnight at +4°C, followed by a further incubation at room temperature for 1h with a goat secondary antibody to Rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

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  Immunocytochemistry/ Immunofluorescence - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Immunofluorescence staining of HeLa cells with purified ab129195 at a working dilution of 1/100, counter-stained with DAPI. The secondary antibody was Alexa Fluor^® 488 goat anti-rabbit (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077), used at a dilution of 1/1000. Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (Alexa Fluor^® 594 goat anti-mouse, 1/1000), shown in the top right hand panel. The cells were fixed in 4% PFA and permeabilized using 0.1% Triton X 100. The negative controls are shown in bottom middle and right hand panels - for negative control 1, purified ab129195 was used at a dilution of 1/500 followed by an Alexa Fluor^® 594 goat anti-mouse antibody (Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120) at a dilution of 1/500. For negative control 2, Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at a dilution of 1/400.

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  Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Blocking/diluting buffer and concentration: 5% NFDM/TBST

  All lanes: ab129195 at 1/1000 dilution

  Lane 1: Mouse brain lysates at 20 µg

  Lane 2: Mouse heart lysates at 20 µg

  Lane 3: Mouse liver lysates at 20 µg

  Lane 4: Mouse spleen lysates at 20 µg

  Secondary

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

  Predicted band size: 92 kDa

  Observed band size: 110 kDa

  Exposure time: 180s

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  Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Blocking/diluting buffer and concentration: 5% NFDM/TBST
  

  Observed band: 110kd

  All lanes: ab129195 at 1/1000 dilution

  Lane 1: Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 20 µg

  Lane 2: HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 3: NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates at 20 µg

  Lane 4: C2C12 (Mouse myoblasts myoblast) whole cell lysates at 20 µg

  Lane 5: Mouse skeletal muscle lysates at 20 µg

  Lane 6: C6 (Rat glial tumor glial cell) whole cell lysates at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated at 1/20000 dilution

  Predicted band size: 92 kDa

  Exposure time: 20s

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  Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Lanes 1 - 4: Merged signal (red and green). Green - ab129195 observed at 110 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - Loading Control ab8245, observed at 37 kDa.
  
  ab129195 was shown to specifically react with KMD1 / LSD1 in wild-type HAP1 cells. No band was observed when KMD1 / LSD1 knockout samples were used. Wild-type and KMD1 / LSD1 knockout samples were subjected to SDS-PAGE. ab129195 and Anti-GAPDH antibody [6C5] - Loading Control ab8245 (loading control to GAPDH) were both diluted 1/10,000 and incubated overnight at 4°C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) secondary antibodies at 1/10,000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195) at 1/10000 dilution

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: KMD1 / LSD1 knockout HAP1 cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: Jurkat cell lysate at 20 µg

  Predicted band size: 92 kDa

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  Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195) at 1/10000 dilution

  Lane 1: C6 whole cell lysate at 10 µg

  Lane 2: Raw 264.7 whole cell lysate at 10 µg

  Lane 3: PC-12 whole cell lysate at 10 µg

  Lane 4: NIH/3T3 whole cell lysate at 10 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: 92 kDa

  Observed band size: 110 kDa

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  Flow Cytometry (Intracellular) - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labeling KDM1/LSD1 with purified ab129195 at 1/20 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^® 488) (1/2000 dilution) was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) were used as the unlabeled control.
  

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Immunohistochemical staining of paraffin embedded mouse colon with purified ab129195 at a working dilution of 1/50. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.
  

  PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

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  Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Blocking/Dilution buffer: 5% NFDM/TBST.

  All lanes: Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195) at 1/10000 dilution

  Lane 1: HEK293 whole cell lysate at 10 µg

  Lane 2: HeLa whole cell lysate at 10 µg

  Lane 3: Jurkat whole cell lysate at 10 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: 92 kDa

  Observed band size: 110 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Immunohistochemical staining of paraffin embedded human stomach carcinoma with purified ab129195 at a working dilution of 1/50. The secondary antibody used is Goat Anti-Rabbit IgG H&L (HRP) ab97051, a goat anti-rabbit IgG (H&L) at a dilution of 1/500. The sample is counter-stained with hematoxylin. Antigen retrieval was perfomed using Tris-EDTA buffer, pH 9.0.
  

  PBS was used instead of the primary antibody as the negative control, and is shown in the inset.

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  This image is courtesy of an anonymous customer review

  Immunocytochemistry/ Immunofluorescence - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Unpurified ab129195 staining KDM1/LSD1 in paraffin-embedded A549 lung cancer cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed using the HOPE technique and permeabilized with 0.05% Tween. Samples were incubated with primary antibody (1/100) for 45 minutes at 25°C. An Alexa Fluor^®488-conjugated Donkey anti-mouse IgG polyclonal (1/200) was used as the secondary antibody.

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  Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  All lanes: Western blot - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195) at 1/10000 dilution

  Lane 1: 293T cell lysate at 10 µg

  Lane 2: HeLa cell lysate at 10 µg

  Lane 3: Jurkat cell lysate at 10 µg

  Lane 4: PC3 cell lysate at 10 µg

  Lane 5: C6 cell lysate at 10 µg

  Lane 6: RAW264.7 cell lysate at 10 µg

  Lane 7: PC12 cell lysate at 10 µg

  Lane 8: NIH 3T3 cell lysate at 10 µg

  Secondary

  All lanes: Goat anti-Rabbit HRP at 1/2000 dilution

  Predicted band size: 92 kDa

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  ChIC/CUT&RUN sequencing - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  CUT&RUN was performed using the ChIC/CUT&RUN pAG-MNAse ChIC/CUT&RUN pAG-MNase ab285373 2.5 x 10^5 HeLa cells and 5μg of ab129195 [EPR6825]. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.
  Additional screenshots of mapped reads can be downloaded here.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  Unpurified ab129195, at 1/100, staining KDM1/LSD1 in paraffin embedded human testis tissue by Immunohistochemistry.
  

  Perform heat mediated antigen retrieval before commencing with IHC staining protocol.

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  ChIC/CUT&RUN sequencing - Anti-KDM1/LSD1 antibody [EPR6825] - Nuclear Marker and ChIP Grade (ab129195)

  ChIC/CUT&RUN was performed using a pAG-MNAse at a final concentration of 700 ng/mL. 2.5X10^5 of Human wild-type HeLa cell line (ab255928) or KDM1A (LSD1) knockout HeLa cell line (Human KDM1A (LSD1) knockout HeLa cell line ab265790) were used along with 5µg of ab129195 [EPR6825]. Assay Quality Control was conducted using 5µg Anti-CTCF(Anti-CTCF antibody [EPR18253] - ChIP Grade ab188408) on the same cell lines. The resulting DNA was sequenced on the Illumina NovaSeq 6000 to a depth of 10 million reads.? The negative IgG control Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730 is also shown.
  
  Additional screenshots of mapped reads can be found in the Protocol booklet in the Support and downloads section.
  
  The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.