Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human testis tissue labelling YTHDC1 with ab259990 at 1/500 (1.152 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human testis. The section was incubated with ab220159 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized HeLa (human cervix adenocarcinoma epithelial cell) cells labelling YTHDC1 with ab259990 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HeLa cells labelling YTHDC1 with ab259990 at 1/50 (11.52 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing mainly nuclear staining in HeLa cells (PMID : 18669619) is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized NIH/3T3 (mouse embryonic fibroblast) cells labelling YTHDC1 with ab259990 at 1/500 dilution (0.1ug) (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat anti rabbit IgG (Alexa Fluor^® 488, ab150077) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labelling YTHDC1 with ab259990 at 1/500 (1.152 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat testis. The section was incubated with ab220159 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized NIH/3T3 cells labelling YTHDC1 with ab259990 at 1/50 (11.52 ug/ml) dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing mainly nuclear staining in NIH/3T3 cells (PMID : 18669619) is observed.

ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor^® 594) was used to counterstain tubulin at 1/200 dilution (Red). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) at 1/1000 dilution.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labelling YTHDC1 with ab259990 at 1/500 (1.152 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse testis. The section was incubated with ab220159 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND^® RX instrument. Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins

• WB

Lab

Western blot - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

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

Lysates were made freshly and used in WB test immediately to minimize protein degradation.

Exposure time : 15 seconds

All lanes:

Western blot - Anti-YTHDC1 antibody [EPR21821-213] (<a href='/en-us/products/primary-antibodies/ythdc1-antibody-epr21821-213-ab259990'>ab259990</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 3:

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

Lane 4:

PC-12 (rat adrenal gland pheochromocytoma) 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 1/20000 dilution

Predicted band size: 85 kDa

Observed band size: 100 kDa

false

• WB

Lab

Western blot - Anti-YTHDC1 antibody [EPR21821-213] - BSA and Azide free (AB282289)

This data was developed using ab259990, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS

Lanes 1-4 : Merged signal (red and green). Green - ab259990 observed at 100 kDa. Red - loading control ab8245 observed at 36 kDa.

ab259990 Anti-YTHDC1 antibody [EPR21821-213] was shown to specifically react with YTHDC1 in wild-type ES-D3 cells. Loss of signal was observed when a knockout cell line was used. Wild-type and YTHDC1 knockout samples were subjected to SDS-PAGE. ab259990 and Anti-GAPDH antibody [6C5] - Loading Control (ab8245) were incubated at room temperature for 2. 5 hours at 1 in 1000 dilution and 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.

The wild-type and YTHDC1 knockout ES-D3 lysates were kindly provided by an anonymous collaborator.

All lanes:

Western blot - Anti-YTHDC1 antibody [EPR21821-213] (<a href='/en-us/products/primary-antibodies/ythdc1-antibody-epr21821-213-ab259990'>ab259990</a>) at 1/1000 dilution

Lane 1:

Wild-type ES-D3 (mouse embryonic pluripotent stem Cell) whole cell lysate at 20 µg

Lane 2:

YTHDC1 knockout ES-D3 cell lysate at 20 µg

Lane 3:

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

Lane 4:

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

Secondary

All lanes:

Goat Anti-Rabbit IgG H&L (IRDye® 800CW) (<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) (<a href='/en-us/products/secondary-antibodies/goat-mouse-igg-h-l-irdye-680rd-preadsorbed-ab216776'>ab216776</a>) at 1/10000 dilution

Predicted band size: 85 kDa

Observed band size: 100 kDa

false