Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using ab194299, the same antibody clone in a different buffer formulation. Immunohistochemical analysis of paraffin-embedded human tonsil tissue labeling EHMT2/G9A + EHMT1/GLP with ab194299 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) (ab97051) at 1/500 dilution. Nuclear staining on human tonsil 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.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using ab194299, the same antibody clone in a different buffer formulation.

Intracellular Flow Cytometry analysis of HeLa (human cervix adenocarcinoma) cells labelling EHMT2/G9A + EHMT1/GLP (red) with purified ab194299 at a dilution of 1/150. Goat anti rabbit IgG (Alexa Fluor^® 488) was used as the secondary antibody at 1/2000. Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. Isotype control antibody was Rabbit monoclonal IgG (black). The blue line shows cells without incubation with primary antibody and secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using ab194299, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized HeLa (Human epithelial cell line from cervix adenocarcinoma) cells labeling EHMT2/G9A + EHMT1/GLP with ab194299 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on HeLa cell line. The nuclear counter stain is DAPI (blue). Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red). The negative controls are as follows :
-ve control 1 : ab194299 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) at 1/1000 dilution.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using ab194299, the same antibody clone in a different buffer formulation.Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% tritonX-100 permeabilized NIH/3T3 (Mouse embryonic fibroblast cell line) cells labeling EHMT1/GLP + EHMT2/G9A with ab194299 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG (Alexa Fluor^® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing nuclear staining on NIH/3T3. The nuclear counter stain is DAPI (blue).Tubulin is detected with Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary antibody at 1/1000 dilution (red).
The negative controls are as follows :
-ve control 1 : ab194299 at 1/1000 dilution, followed by Goat Anti-Mouse IgG H&L (Alexa Fluor^® 594) (ab150120) secondary at 1/1000 dilution.
-ve control 2 : Anti-alpha Tubulin mouse MAb (ab7291) at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) (ab150077) secondary at 1/1000 dilution.

• WB

Lab

Western blot - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using the same antibody clone in a different buffer formulation (ab194299 ).

Western blot : Anti-EHMT1 antibody [EPR18667] (ab194299) staining at 1/1000 dilution, shown in green; Mouse anti-CANX [CANX/1543] (ab238078) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab194299 was shown to bind specifically to EHMT1. A band was observed at 165 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in EHMT1 knockout cell line. To generate this image, wild-type and EHMT1 knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-EHMT2/G9A + EHMT1/GLP antibody [EPR18667] (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-ehmt1-glp-antibody-epr18667-ab194299'>ab194299</a>) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

EHMT1 knockout HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human EHMT1 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-ehmt1-knockout-hct116-cell-line-ab287388'>ab287388</a>)

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 165 kDa

false

• WB

Supplier Data

Western blot - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using ab194299, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-EHMT2/G9A + EHMT1/GLP antibody [EPR18667] (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-ehmt1-glp-antibody-epr18667-ab194299'>ab194299</a>) at 1/1000 dilution

All lanes:

Human fetal kidney lysate at 10 µg

Secondary

All lanes:

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

Predicted band size: 132 kDa,141 kDa

Observed band size: 170 kDa

true

Exposure time: 3min

• WB

Supplier Data

Western blot - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using ab194299, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

Exposure times : Lanes 1-2 : 20 seconds; Lane 3 : 30 seconds.

All lanes:

Western blot - Anti-EHMT2/G9A + EHMT1/GLP antibody [EPR18667] (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-ehmt1-glp-antibody-epr18667-ab194299'>ab194299</a>) at 1/5000 dilution

Lane 1:

HEK-293 (Human epithelial cell line from embryonic kidney) whole cell lysate at 10 µg

Lane 2:

HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell lysate at 10 µg

Lane 3:

K562 (Human chronic myelogenous leukemia cell line from bone marrow) 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 1/100000 dilution

Predicted band size: 132 kDa,136 kDa,141 kDa

Observed band size: 136 kDa,170 kDa

false

• WB

Supplier Data

Western blot - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

Blocking buffer and concentration : 5% NFDM/TBST  Diluting buffer and concentration : 5% NFDM/TBST Exposure Time : Lane 1 : 15 seconds, Lane 2-4 : 37 seconds. Lysates were freshly made and used for Western blotting immediately to minimize protein degradation. This data was developed using ab194299, the same antibody clone in a different buffer formulation.

All lanes:

Western blot - Anti-EHMT2/G9A + EHMT1/GLP antibody [EPR18667] (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-ehmt1-glp-antibody-epr18667-ab194299'>ab194299</a>) at 1/1000 dilution

Lane 1:

HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

NIH/3T3 (mouse embryonic fibroblast) whole cell lysate at 20 µg

Lane 3:

PC-12 (rat adrenal gland pheochromocytoma cell) whole cell lysate at 20 µg

Lane 4:

Raw 264.7 (mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 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 1/100000 dilution

Predicted band size: 141 kDa,132 kDa

Observed band size: 170 kDa

false

• WB

Lab

Western blot - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

False colour image of Western blot : Anti-EHMT2/G9A + EHMT1/GLP antibody [EPR18667] staining at 1/1000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab194299 was shown to bind specifically to EHMT1/GLP and EHMT2/G9A. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4°C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times, then imaged. Secondary antibodies used were Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-EHMT2/G9A + EHMT1/GLP antibody [EPR18667] (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-ehmt1-glp-antibody-epr18667-ab194299'>ab194299</a>) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 40 µg

Lane 2:

EHMT1 knockout HAP1 cell lysate at 40 µg

Lane 3:

Wild-type HAP1 Nuclear cell lysate at 40 µg

Lane 4:

HeLa cell lysate at 40 µg

Lane 5:

HeLa Nuclear cell lysate at 40 µg

Lane 6:

Wild-type HEK-293T cell lysate at 40 µg

Lane 7:

Wild-type HEK-293T nuclear cell lysate at 40 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L (IRDye® 800CW) preabsorbed (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-irdye-800cw-preadsorbed-ab216773'>ab216773</a>) and Goat anti-Mouse IgG H&L (IRDye®680RD) preabsorbed (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/20000 dilution

Predicted band size: 132 kDa,141 kDa

Observed band size: 150-170 kDa

false

• WB

Supplier Data

Western blot - Anti-EHMT2/G9A+EHMT1/GLP antibody [EPR18667] - BSA and Azide free (AB251187)

This data was developed using ab194299, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer : 5% NFDM/TBST.

EHMT2 recombinant protein fragment with His-Tag^®.

Lane 1:

Western blot - Anti-EHMT2/G9A + EHMT1/GLP antibody [EPR18667] (<a href='/en-us/products/primary-antibodies/ehmt2-g9a-ehmt1-glp-antibody-epr18667-ab194299'>ab194299</a>) at 1/5000 dilution

Lane 2:

Anti His-Tag®.

All lanes:

EHMT2 recombinant protein at 0.01 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 1/100000 dilution

Predicted band size: 132 kDa,141 kDa

Observed band size: 30 kDa,60 kDa

true

Exposure time: 5s