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

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : MECP2 knockout HAP1 cell lysate
Lane 3 : SH-SY5Y cell lysate
Lane 4 : Mouse Brain cell lysate
Lane 5 : HepG2 cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 52 kDa
Observed band size: 68-70 kDa why is the actual band size different from the predicted?

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.

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.

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

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.

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

Lane 1 : Human brain tissue lysate
Lane 2 : Human hippocampus tissue lysate
Lane 3 : Human liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

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

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse hippocampus tissue lysate
Lane 3 : Neuro-2a (mouse neuroblastoma neuroblast), whole cell lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

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

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

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

Lane 1 : Rat brain tissue lysate
Lane 2 : Rat liver tissue lysate

Lysates/proteins at 20 µg per lane.

Secondary
All lanes : Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

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

Anti-MeCP2 antibody [EPR23201-3] (ab253197) at 1/1000 dilution + SH-SY5Y (human neuroblastoma epithelial cell), whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (ab97051) at 1/100000 dilution

Predicted band size: 52 kDa
Observed band size: 75 kDa why is the actual band size different from the predicted?

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

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.

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

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

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

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

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

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.