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

Lane 1 : Wild-type HeLa cell lysate
Lane 2 : KDM1A knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

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.

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.

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).

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.

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.

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.

All lanes : ab129195 at 1/1000 dilution

Lane 1 : Mouse brain lysates
Lane 2 : Mouse heart lysates
Lane 3 : Mouse liver lysates
Lane 4 : Mouse spleen lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?


Exposure time: 180 seconds

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

 

All lanes : ab129195 at 1/1000 dilution

Lane 1 : Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates
Lane 2 : HCT 116 (Human colorectal carcinoma epithelial cell) whole cell lysates
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast) whole cell lysates
Lane 4 : C2C12 (Mouse myoblasts myoblast) whole cell lysates
Lane 5 : Mouse skeletal muscle lysates
Lane 6 : C6 (Rat glial tumor glial cell) whole cell lysates

Lysates/proteins at 20 µg per lane.

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

Predicted band size: 92 kDa


Exposure time: 20 seconds

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

Observed band: 110kd

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

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : KMD1 / LSD1 knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Jurkat cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 92 kDa

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

Lane 1 : C6 whole cell lysate
Lane 2 : Raw 264.7 whole cell lysate
Lane 3 : PC-12 whole cell lysate
Lane 4 : NIH/3T3 whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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 Fluor^® 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.

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.

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

Lane 1 : HEK293 whole cell lysate
Lane 2 : HeLa whole cell lysate
Lane 3 : Jurkat whole cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 92 kDa
Observed band size: 110 kDa why is the actual band size different from the predicted?

Blocking/Dilution buffer: 5% NFDM/TBST.

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.

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.

See Abreview

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

Lane 1 : 293T cell lysate
Lane 2 : HeLa cell lysate
Lane 3 : Jurkat cell lysate
Lane 4 : PC3 cell lysate
Lane 5 : C6 cell lysate
Lane 6 : RAW264.7 cell lysate
Lane 7 : PC12 cell lysate
Lane 8 : NIH 3T3 cell lysate

Lysates/proteins at 10 µg per lane.

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

Predicted band size: 92 kDa

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.