All lanes : Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : FMRP knockout HAP1 cell lysate
Lane 3 : HeLa cell lysate
Lane 4 : Human brain cell lysate

Lysates/proteins at 20 µg per lane.

Performed under reducing conditions.

Predicted band size: 69 kDa
Observed band size: 77 kDa why is the actual band size different from the predicted?

This data was developed using the same antibody clone in a different buffer formulation (ab259335).

Lanes 1 - 4: Merged signal (red and green). Green - ab259335 observed at 77 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37 kDa.

ab259335 was shown to react with FMRP in western blot. The band observed in the knockout lysate lane below 77 kDa is likely to represent a truncated form. This has not been investigated further. Membranes were blocked in 3 % milk in TBS-T (0.1 % Tween^®) before incubation with ab259335 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 h at room temperature before imaging.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse cerebrum (PMID: 24463622). The section was incubated with ab259335 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.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized C2C12 cells labelling FMRP with ab259335 at 1/50 dilution, followed by ab150077 Goat Anti-Rabbit IgG H&L (Alexa Fluor^® 488) antibody at 1/1000 dilution (Green). Confocal image showing strong cytoplasmic and weak nuclear staining in C2C12 cells. 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.

All lanes : Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse hippocampus tissue lysate
Lane 3 : Rat hippocampus 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: 69 kDa
Observed band size: 75,80 kDa why is the actual band size different from the predicted?

This data was developed using ab259335, the same antibody clone in a different buffer formulation. 

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

Multiple bands could be seen due to alternative splicing of FMRP.

Exposure time: 180 seconds.

All lanes : Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

Lane 1 : HeLa (human cervix adenocarcinoma epithelial cell) whole cell lysate
Lane 2 : NIH/3T3 (mouse embryonic fibroblast), whole cell lysate
Lane 3 : PC-12 (rat adrenal gland pheochromocytoma), 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: 69 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

This data was developed using ab259335, the same antibody clone in a different buffer formulation. 

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

Exposure time: 26 seconds.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Flow cytometric analysis of 4% paraformaldehyde-fixed, 90% methanol-permeabilized C2C12 (Mouse myoblast) cells labelling FMRP with ab259335 at 1/500 dilution (Red) compared with a Rabbit monoclonal IgG (ab172730)  isotype control (Black) 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.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

FMRP was immunoprecipitated from 0.35 mg C2C12 (Mouse myoblast) whole cell lysate 10ug with ab259335 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259335 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: C2C12 (Mouse myoblast) whole cell lysate 10ug

Lane 2: ab259335 IP in C2C12 whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259335 in C2C12 whole cell lysate

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

Exposure time: 10 seconds.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse testis tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on mouse testis (PMID: 16790844). The section was incubated with ab259335 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.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat cerebrum tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat cerebrum. The section was incubated with ab259335 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.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Rat testis tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Positive staining on rat testis. The section was incubated with ab259335 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.

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

FMRP was immunoprecipitated from 0.35 mg HeLa (human cervix adenocarcinoma epithelial cell ) whole cell lysate with ab259335 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab259335 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP)(ab131366) was used at 1/5000 dilution.

Lane 1: HeLa (human cervix adenocarcinoma epithelial cell ) whole cell lysate 10ug

Lane 2: ab259335 IP in HeLa whole cell lysate

Lane 3: Rabbit monoclonal IgG (ab172730) instead of ab259335 in HeLa whole cell lysate

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

Exposure time: 10 seconds.

All lanes : Anti-FMRP antibody [EPR23852-90] (ab259335) at 1/1000 dilution

Lane 1 : Mouse brain tissue lysate
Lane 2 : Mouse heart 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: 69 kDa
Observed band size: 75,80 kDa why is the actual band size different from the predicted?

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Blocking and dilution buffer: 5% NFDM/TBST.

Exposure time: 81 seconds.

Negative control: Mouse heart(PMID: 7633436).

Multiple bands could be seen due to alternative splicing of FMRP

This data was developed using ab259335, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded mouse cardiac muscle tissue labeling FMRP with ab259335 at 1/10000 (0.04 ug/ml) followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (ab209101). Negative control: No staining on mouse cardiac muscle (PMID: 7633436). The section was incubated with ab259335 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.