All lanes : Anti-FMRP antibody (ab17722) at 1 µg/ml

Lane 1 : Wild-type HAP1 cell lysate
Lane 2 : FMR1 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: 71 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?

Lanes 1 - 4: Merged signal (red and green). Green - ab17722 observed at 80 kDa. Red - loading control, ab8245 (Mouse anti-GAPDH antibody [6C5]) observed at 37kDa.

ab17722 was shown to react with FMR1 in HAP1 wild-type cells in western blot. Loss of signal was observed when FMR1 knockout sample was used. HAP1 wild-type and FMR1 knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 3% milk in TBS-T (0.1% Tween^®) before incubation with ab17722 and ab8245 (Mouse anti-GAPDH antibody [6C5]) overnight at 4°C at 1 µg/ml 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.

ab17722 stained in SK-N-SH (Human neuroblastoma cell line) cells.

Cells were fixed with 100% methanol (5 min) at room temperature and incubated with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% triton for 1h at room temperature to permeabilize the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab17722 at 5 µg/ml and ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were ab150120 (pseudo-colored red) and ab150081 (colored green) used at 1 µg/ml for 1hour at room temperature. DAPI was used to stain the cell nuclei (colored blue) at a concentration of 1.43µM for 1hour at room temperature.

IHC image of FMRP staining in mouse frontal cortex section.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6). The section was then blocked using 1% BSA for 10 mins at 21°C The section was then incubated with ab17722, 1/1500, for 2 hours at 21°C. The section was then counterstained with hematoxylin.

See Abreview

The specificity of the C-terminal, phospho-insensitive FMRP tFMRP antibody (Abcam ab17722) was verified by immunoblotting whole cell cortical lysates from 2 month-old Fmr1^y/+ (WT) and Fmr1^y/− (KO) mice. Short and long exposures (exp.) are shown. Arrows point to three FMRP-specific bands while asterisks point to nonspecific bands.

Anti-FMRP antibody (ab17722) at 1 µg/ml + Brain (Mouse) Tissue Lysate at 10 µg

Secondary
Goat polyclonal to Rabbit IgG - H&L - Pre-Adsorbed (HRP) at 1/3000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?
Additional bands at: 105 kDa, 23 kDa, 75 kDa (possible isoform). We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

FMRP was immunoprecipitated using 0.5mg HeLa (Human epithelial cell line from cervix adenocarcinoma) whole cell extract, 5µg of Rabbit polyclonal to FMRP and 50µl of protein G magnetic beads (+). No antibody was added to the control (-).
The antibody was incubated under agitation with Protein G beads for 10min, HeLa whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.
Proteins were eluted by addition of 40µl SDS loading buffer and incubated for 10min at 70^oC; 10µl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab17722.
Secondary: Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).
Band: 80kDa: FMRP.

All lanes : Anti-FMRP antibody (ab17722) at 1 µg/ml

Lane 1 : HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate
Lane 2 : HeLa (Human epithelial carcinoma cell line) Nuclear Lysate

Lysates/proteins at 10 µg per lane.

Secondary
All lanes : IRDye 680 Conjugated Goat Anti-Rabbit IgG (H+L) at 1/10000 dilution

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?
Additional bands at: 75 kDa (possible isoform)

Anti-FMRP antibody (ab17722) at 1 µg/ml + PC-12 (Rat adrenal pheochromocytoma cell line) whole cell lysate at 20 µg

Secondary
Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (ab97080) at 1/5000 dilution

Developed using the ECL technique.

Performed under reducing conditions.

Predicted band size: 71 kDa
Observed band size: 80 kDa why is the actual band size different from the predicted?
Additional bands at: 13 kDa, 32 kDa, 68 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 2 minutes

FMRP is localized at various intracellular sites in HeLa cells.

Confocal laser scanning microscopy (cLSM) images of HeLa (Human epithelial cell line from cervix adenocarcinoma) cells depicting endogenous FMRP (ab17722, green channel) costained with various cytosolic (Panel A, not shown) and nuclear (Panel B, shown) markers (red channel). DNA was stained by using DAPI (blue channel). Boxed areas in the merged panels depict enlarged areas of interest. Scale bar: 10 μm.

HeLa cells grown on glass coverslips were fixed with 4% paraformaldehyde for 20 min, permeabilized with 0.25% triton X-100 in PBS for 10 min, and thereafter blocked for 1 h in a solution containing 3% BSA in 0.25% triton-X100/PBS. Cells were incubated with primary and secondary antibodies for 1 h and finally counterstained with DAPI for 5 min and mounted using the prolong gold anti-fade reagent.

ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (R,S)-3,5-DHPG (ab120020), by ICC/IF. Increase in FMRP expression correlates with increased concentration of (R,S)-3,5-DHPG, as described in literature.
The cells were incubated at 37°C for 1h in media containing different concentrations of ab120020 ((R,S)-3,5-DHPG) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab17722 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (R,S)-MCPG sodium salt (ab120252), by ICC/IF. Decrease of FMRP expression correlates with increased concentration of (R,S)-MCPG sodium salt, as described in literature.
The cells were incubated at 37°C for 2h in media containing different concentrations of ab120252 ((R,S)-MCPG sodium salt) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab17722 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (S)-MCPG (ab120059), by ICC/IF. Decrease of FMRP expression correlates with increased concentration of (S)-MCPG, as described in literature.
The cells were incubated at 37°C for 30 minutes in media containing different concentrations of ab120059 ((S)-MCPG) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab17722 (5 μg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (R,S)-MCPG (ab120033), by ICC/IF. Decrease in FMRP expression correlates with increased concentration of (R,S)-MCPG, as described in literature.
The cells were incubated at 37°C for 2h in media containing different concentrations of ab120033 ((R,S)-MCPG) in DMSO, fixed with 4% formaldehyde for 10 minutes at room temperature and blocked with PBS containing 10% goat serum, 0.3 M glycine, 1% BSA and 0.1% tween for 2h at room temperature. Staining of the treated cells with ab17722 (5 µg/ml) was performed overnight at 4°C in PBS containing 1% BSA and 0.1% tween. A DyLight 488 goat anti-rabbit polyclonal antibody (ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

IHC image of FMRP staining in human tonsil FFPE section, performed on a Bond^TM system using the standard protocol F.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20 mins. The section was then incubated with ab17722, 5µg/ml, for 15 mins at room temperature and detected using an HRP conjugated compact polymer system. DAB was used as the chromogen. The section was then counterstained with hematoxylin and mounted with DPX.