Anti-MeCP2 antibody [EPR23201-3]

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunohistochemical analysis of paraffin-embedded Human astrocytoma tissue labeling MeCP2 with ab253197 at 1/4000 dilution (0.158 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human astrocytoma (PMID : 11809720). The section was incubated with ab253197 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized SH-SY5Y cells labelling MeCP2 with ab253197 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing nuclear staining in SH-SY5Y cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 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 1000 2 ug/ml dilution.

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized SH-SY5Y (Human neuroblastoma epithelial cell) cells labelling MeCP2 with ab253197 at 1/600 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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labeling MeCP2 with ab253197 at 1/4000 dilution (0.158 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on human kidney (PMID : 11809720). The section was incubated with ab253197 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

MeCP2 was immunoprecipitated from 0.35 mg of 293T (human embryonic kidney epithelial cell), whole cell lysate with ab253197 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab253197 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution. Lane 1 : 293T (human embryonic kidney epithelial cell), whole cell lysate 10 μg (Input). Lane 2 : ab253197 IP in 293T whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab253197 in 293T whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 58 seconds.

All lanes:

Immunoprecipitation - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

All lanes:

293T (human embryonic kidney epithelial cell), whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 58s

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunohistochemical analysis of paraffin-embedded Mouse lung tissue labeling MeCP2 with ab253197 at 1/4000 dilution (0.158 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse lung (PMID : 11809720). The section was incubated with ab253197 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• ICC/IF

Unknown

Immunocytochemistry/ Immunofluorescence - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Neuro-2a cells labelling MeCP2 with ab253197 at 1/500 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2 ug/ml dilution (Green). Confocal image showing nuclear staining in Neuro-2a cells. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5 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 1000 2 ug/ml dilution.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling MeCP2 with ab253197 at 1/4000 dilution (0.158 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on mouse testis (PMID : 11809720). The section was incubated with ab253197 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• Flow Cyt (Intra)

Unknown

Flow Cytometry (Intracellular) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Neuro-2a (Mouse neuroblastoma neuroblast) cells labelling MeCP2 with ab253197 at 1/600 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

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling MeCP2 with ab253197 at 1/4000 dilution (0.158 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat cerebrum (PMID : 11809720). The section was incubated with ab253197 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Immunohistochemical analysis of paraffin-embedded Rat lung tissue labeling MeCP2 with ab253197 at 1/4000 dilution (0.158 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Nuclear staining on rat lung (PMID : 11809720). The section was incubated with ab253197 for 10 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 Citrate buffer (pH 6.0, epitope retrieval solution 1) for 20 mins

• IP

Supplier Data

Immunoprecipitation - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

MeCP2 was immunoprecipitated from 0.35 mg of Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate with ab253197 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab253197 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5000 dilution. Lane 1 : Neuro-2a whole cell lysate 10 μg (Input). Lane 2 : ab253197 IP in Neuro-2a whole cell lysate. Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab253197 in Neuro-2a whole cell lysate. Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 58 seconds.

All lanes:

Immunoprecipitation - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

All lanes:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 58s

• WB

Lab

Western blot - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Lanes 1 - 5 : Merged signal (red and green). Green - ab253197 observed at 68-70 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab253197 was shown to react with MeCP2 in wild-type HAP1 cells in western blot. Loss of signal was observed when MECP2 knockout sample was used. Wild-type and MECP2 knockout HAP1 cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBS-T (0.1% Tween^®) before incubation with ab253197 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at a 1 in 1000 dilution and a 1 in 20000 dilution respectively. Blots were incubated 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 in 20000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

Lane 1:

Wild-type HAP1 cell lysate at 20 µg

Lane 2:

MECP2 knockout HAP1 cell lysate at 20 µg

Lane 3:

SH-SY5Y cell lysate at 20 µg

Lane 4:

Mouse Brain cell lysate at 20 µg

Lane 5:

HepG2 cell lysate at 20 µg

Predicted band size: 52 kDa

Observed band size: 68-70 kDa

false

• WB

Lab

Western blot - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Blocking and diluting buffer and concentration : 5% NFDM/TBST This blot was developed using a higher sensitivity ECL substrate.

The lower bands below 75kDa should be degraded MeCP2 fragments(PMID : 31601272), freshly prepared lysates were recommended to use in avoid of target protein degradation.

Exposure time : 3 minutes

All lanes:

Western blot - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

Lane 1:

Human brain tissue lysate at 20 µg

Lane 2:

Human hippocampus tissue lysate at 20 µg

Lane 3:

Human liver tissue lysate 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 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

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

This antibody specifically recognizes MeCP2 protein at around 75kDa (PMID : 27995568, PMID : 28394263)

Exposure time : 114 seconds

All lanes:

Western blot - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

All lanes:

SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate 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 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

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

Exposure time : Lane1-2 : 30 seconds Lane 3 : 3 minutes

All lanes:

Western blot - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse hippocampus tissue lysate at 20 µg

Lane 3:

Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate 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 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 75 kDa

false

• WB

Lab

Western blot - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Western blot : Rabbit Monoclonal[EPR23201-3] to MeCP2 ab253197 staining at 1/1000 dilution, shown in green; Mouse anti GAPDH (ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 52 kDa in Wild-type HCT 116 ab288559 cell lysates with no signal observed at this size in MECP2 knockout HCT 116 ab289193 cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5pc 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 800CW & Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

Lane 1:

Wild-type HCT 116 ab288559 at 20 µg

Lane 2:

Western blot - Human MECP2 knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-mecp2-knockout-hct116-cell-line-ab289193'>ab289193</a>) at 20 µg

Lane 3:

Wild-type HAP1 at 20 µg

Lane 4:

MECP2 knockout HAP1 at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 52 kDa

Observed band size: 52 kDa

false

• WB

Lab

Western blot - Anti-MeCP2 antibody [EPR23201-3] (AB253197)

Blocking and diluting buffer and concentration : 5% NFDM/TBST Exposure time : 3 minutes

All lanes:

Western blot - Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution

Lane 1:

Rat brain tissue lysate at 20 µg

Lane 2:

Rat liver tissue lysate 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 1/100000 dilution

Predicted band size: 52 kDa

Observed band size: 75 kDa

false