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

• IHC-P

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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.

• ICC/IF

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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 (ab150077), used at a dilution of 1/1000. ab7291, a mouse anti-tubulin antibody (1/1000), was used to stain tubulin along with 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 (ab150120) at a dilution of 1/500. For negative control 2, ab7291 (mouse anti-tubulin) was used at a dilution of 1/500 followed by an Alexa Fluor^® 488 goat anti-rabbit antibody (ab150077) at a dilution of 1/400.

• IHC-P

Unknown

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 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.

• ICC/IF

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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.

This image is courtesy of an anonymous Abreview

• ICC/IF

Lab

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 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) (ab150081) at 2 μg/ml (shown in green). Nuclear DNA was labelled in blue with DAPI.

• Flow Cyt (Intra)

Lab

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.

• IP

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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 (ab172730).

For western blotting, 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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• ChIP

Supplier Data

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).

• IHC-P

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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 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.

• IHC-P

Unknown

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 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.

• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

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• WB

Lab

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 (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 ab265790 (knockout 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 (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 (ab216773) and 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 (<a href='/en-us/products/cell-lines/human-kdm1a-lsd1-knockout-hela-cell-line-ab265790'>ab265790</a>)

Predicted band size: 155 kDa,92 kDa

Observed band size: 110 kDa,160 kDa

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• WB

Lab

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, 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 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 (ab216773) and 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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• WB

Unknown

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

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Exposure time: 20s

• ChIC/CUT&RUN-seq

Supplier Data

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 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.

• ChIC/CUT&RUN-seq

Supplier Data

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 (ab265790) were used along with 5µg of ab129195 [EPR6825]. Assay Quality Control was conducted using 5µg Anti-CTCF(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 ab172730 is also shown. Additional screenshots of mapped reads can be found in the Protocol booklet in the Product Protocol section. The University of Geneva owns patents relevant to ChIC (Chromatin Immuno-Cleavage) methods.

• WB

Lab

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) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

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• WB

Unknown

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 (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 92 kDa

Observed band size: 110 kDa

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Exposure time: 180s

• WB

Unknown

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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