All lanes : Anti-KMT1B / SUV39H2 antibody [EPR18495] (ab190870) at 1/1000 dilution

Lane 1 : Wild-type U2-OS whole cell lysate
Lane 2 : SUV39H2 knockout U2-OS whole cell lysate
Lane 3 : HEK-293 whole cell lysate
Lane 4 : HeLa whole cell lysate

Lysates/proteins at 20 µg per lane.

Predicted band size: 47 kDa
Observed band size: 47 kDa

Lanes 1 - 4: Merged signal (red and green). Green - ab190870 observed at 47 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab190870 was shown to recognize SUV39H2 in wild-type U2-OS cells as signal was lost at the expected MW in SUV39H2 knockout cells. Additional cross-reactive bands were observed in the wild-type and knockout cells. Wild-type and SUV39H2 knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab190870 and ab8245 (Mouse anti-GAPDH loading control) were incubated overnight at 4°C at 1/1000 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.

All lanes : Anti-KMT1B / SUV39H2 antibody [EPR18495] (ab190870) at 1/1000 dilution

Lane 1 : SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate. at 20 µg
Lane 2 : MOLT-4 (Human lymphoblastic leukemia cell line) whole cell lysate at 20 µg
Lane 3 : Human fetal kidney at 10 µg

Secondary
Lanes 1-2 : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution
Lane 3 : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 47 kDa
Observed band size: 47 kDa


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST

All lanes : Anti-KMT1B / SUV39H2 antibody [EPR18495] (ab190870) at 1/10000 dilution

Lane 1 : HEK-293 (Human epithelial cell line from embryonic kidney) cell lysate
Lane 2 : HeLa (Human epithelial cell line from cervix adenocarcinoma) cell lysate
Lane 3 : NIH/3T3 (Mouse embryonic fibroblast cell line) cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/50000 dilution

Developed using the ECL technique.

Predicted band size: 47 kDa
Observed band size: 47,54 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The full length SUV39H2 orthologues differ in size: human 410aa (UniProt Q9H5I1), mouse 477aa (UniProt Q9EQQ0) and rat 481aa (UniProt D3ZIH5).

All lanes : Anti-KMT1B / SUV39H2 antibody [EPR18495] (ab190870) at 1/5000 dilution

Lane 1 : Human testis lysate
Lane 2 : Mouse testis lysate
Lane 3 : Rat testis lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : Anti-Rabbit IgG (HRP), specific to the non-reduced form of IgG at 1/10000 dilution

Developed using the ECL technique.

Predicted band size: 47 kDa
Observed band size: 47,54 kDa why is the actual band size different from the predicted?


Exposure time: 3 minutes

Blocking/Dilution buffer: 5% NFDM/TBST.

The full length SUV39H2 orthologues differ in size: human 410aa (UniProt Q9H5I1), mouse 477aa (UniProt Q9EQQ0) and rat 481aa (UniProt D3ZIH5).

Immunohistochemical analysis of paraffin-embedded human testis tissue labeling KMT1B / SUV39H2 with ab190870 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on germ cells of human testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded mouse testis tissue labeling KMT1B / SUV39H2 with ab190870 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nucleuarstaining on germ cells of mouse testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

Immunohistochemical analysis of paraffin-embedded rat testis tissue labeling KMT1B / SUV39H2 with ab190870 at 1/500 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Nuclear staining on germ cells of rat testis is observed.

Counter stained with Hematoxylin.

Secondary antibody only control: Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution.

Perform heat mediated antigen retrieval with Tris/EDTA buffer pH 9.0 before commencing with IHC staining protocol.

KMT1B / SUV39H2 was immunoprecipitated from 1mg of SH-SY5Y (Human neuroblastoma cell line from bone marrow) whole cell lysate with ab190870 at 1/50 dilution.

Western blot was performed from the immunoprecipitate using ab190870 at 1/1000 dilution.

VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/10000 dilution.

Lane 1: SH-SY5Y whole cell lysate 10µg (Input).

Lane 2: ab190870 IP in SH-SY5Y whole cell lysate.

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab190870 in SH-SY5Y whole cell lysate.

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

Exposure time: 30 seconds.