Anti-FMRP antibody

16 product images

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  Taha et al PLoS One. 2014 Mar 21;9(3):e91465. doi: 10.1371/journal.pone.0091465. eCollection 2014. Fig 1. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Immunocytochemistry/ Immunofluorescence - Anti-FMRP antibody (ab17722)

  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.

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  Bartley et al PLoS One. 2014 May 7;9(5):e96956. doi: 10.1371/journal.pone.0096956. eCollection 2014. Fig 1. Reproduced under the Creative Commons license http://creativecommons.org/licenses/by/4.0/

  Western blot - Anti-FMRP antibody (ab17722)

  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.

  All lanes: Western blot - Anti-FMRP antibody (ab17722)

  Predicted band size: 71 kDa

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  Immunoprecipitation - Anti-FMRP antibody (ab17722)

  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) (Mouse monoclonal [SB62a] Anti-Rabbit IgG light chain (HRP) ab99697).
  Band: 80kDa: FMRP.

  All lanes: Immunoprecipitation - Anti-FMRP antibody (ab17722)

  Predicted band size: 71 kDa

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  Western blot - Anti-FMRP antibody (ab17722)

  Lanes 1 - 4: Merged signal (red and green). Green - ab17722 observed at 80 kDa. Red - loading control, Anti-GAPDH antibody [6C5] - 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 Anti-GAPDH antibody [6C5] - Loading Control 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 (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (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.

  All lanes: Western blot - Anti-FMRP antibody (ab17722) at 1 µg/mL

  Lane 1: Wild-type HAP1 cell lysate at 20 µg

  Lane 2: FMR1 knockout HAP1 cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: Human Brain cell lysate at 20 µg

  Performed under reducing conditions.

  Predicted band size: 71 kDa

  Observed band size: 80 kDa

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  Immunocytochemistry/ Immunofluorescence - Anti-FMRP antibody (ab17722)

  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 Anti-alpha Tubulin antibody [DM1A] - Loading Control ab7291 (Mouse monoclonal [DM1A] to alpha Tubulin - Loading Control) at 1/1000 dilution overnight at +4°C. The secondary antibodies were Goat Anti-Mouse IgG H&L (Alexa Fluor® 594) preadsorbed ab150120 (pseudo-colored red) and Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed 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.

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  This image is courtesy of a customer review submitted by Carl Hobbs, King's College London, United Kingdom

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FMRP antibody (ab17722)

  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.

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  Western blot - Anti-FMRP antibody (ab17722)

  All lanes: Western blot - Anti-FMRP antibody (ab17722) at 1 µg/mL

  All lanes: Brain (Mouse) Tissue Lysate at 10 µg

  Secondary

  All lanes: 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: 105 kDa, 23 kDa, 75 kDa, 80 kDa

  Exposure time: 4min

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  Western blot - Anti-FMRP antibody (ab17722)

  All lanes: Western blot - Anti-FMRP antibody (ab17722) at 1 µg/mL

  Lane 1: HeLa (Human epithelial carcinoma cell line) Whole Cell Lysate at 10 µg

  Lane 2: HeLa (Human epithelial carcinoma cell line) Nuclear Lysate at 10 µg

  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: 75 kDa, 80 kDa

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  Western blot - Anti-FMRP antibody (ab17722)

  All lanes: Western blot - Anti-FMRP antibody (ab17722) at 1 µg/mL

  All lanes: PC-12 (Rat adrenal pheochromocytoma cell line) whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) preadsorbed (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: 13 kDa, 32 kDa, 68 kDa, 80 kDa

  Exposure time: 2min

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  Immunocytochemistry/ Immunofluorescence - Anti-FMRP antibody (ab17722)

  ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (R,S)-MCPG sodium salt ((R,S)-MCPG sodium salt, Group I /II metabotropic glutamate antagonist 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 (R,S)-MCPG sodium salt, Group I /II metabotropic glutamate antagonist 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 (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

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  Immunocytochemistry/ Immunofluorescence - Anti-FMRP antibody (ab17722)

  ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (S)-MCPG ((S)-MCPG, Group I / II mGlu antagonist 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 (S)-MCPG, Group I / II mGlu antagonist 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 (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

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  Immunocytochemistry/ Immunofluorescence - Anti-FMRP antibody (ab17722)

  ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (R,S)-3,5-DHPG ((R,S)-3,5-DHPG, group I mGlu receptor agonist 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 (R,S)-3,5-DHPG, group I mGlu receptor agonist 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 (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

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  Immunocytochemistry/ Immunofluorescence - Anti-FMRP antibody (ab17722)

  ab17722 staining FMRP in SK-N-SH (Human neuroblastoma cell line) cells treated with (R,S)-MCPG ((R,S)-MCPG, Group I /II metabotropic glutamate antagonist 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 (R,S)-MCPG, Group I /II metabotropic glutamate antagonist 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 (Goat Anti-Rabbit IgG H&L (DyLight® 488) preadsorbed ab96899) at 1/250 dilution was used as the secondary antibody. Nuclei were counterstained with DAPI and are shown in blue.

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  Western blot - Anti-FMRP antibody (ab17722)

  False colour image of Western blot: Anti-FMRP antibody staining at 1 ug/ml, shown in green; Mouse anti-GAPDH antibody [6C5] (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab17722 was shown to bind specifically to FMRP. A band was observed at 77 kDa in wild-type A549 cell lysates with no signal observed at this size in Fmr1 knockout cell line. To generate this image, wild-type and Fmr1 knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3% 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

  All lanes: Western blot - Anti-FMRP antibody (ab17722) at 1 µg

  Lane 1: Wild-type A549 cell lysate at 20 µg

  Lane 2: Fmr1 knockout A549 cell lysate at 20 µg

  Lane 3: HeLa cell lysate at 20 µg

  Lane 4: Human Brain cell lysate at 20 µg

  Performed under reducing conditions.

  Observed band size: 77 kDa

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  Lab

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FMRP antibody (ab17722)

  IHC image of FMRP staining in a section of formalin-fixed paraffin-embedded human normal cerebral cortex* performed on a Leica Biosystems BOND^® RX instrument. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH6, epitope retrieval solution 1) for 20mins. The section was then incubated with ab17722, 5ug/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 haematoxylin and mounted with DPX. The inset secondary-only control image is taken from an identical assay without primary antibody.
  
  For other IHC staining systems (automated and non-automated) customers should optimize variable parameters such as antigen retrieval conditions, primary antibody concentration and antibody incubation times.
  
  *Tissue obtained from the Human Research Tissue Bank, supported by the NIHR Cambridge Biomedical Research Centre."

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  Western blot - Anti-FMRP antibody (ab17722)

  Western blot: Anti-FMRP antibody ab17722 staining at 1 µg/mL, shown in green; Mouse anti GAPDH (Anti-GAPDH antibody [6C5] - Loading Control ab8245) loading control staining at 1/20,000 dilution, shown in magenta. A band was observed at 75 kDa in Wild-type U-87 MG cell lysates with no signal observed at this size in FMR1 knockout U-87 MG cell line. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-FMRP antibody (ab17722) at 1 µg/mL

  Lane 1: Wild-type U-87 MG at 20 µg

  Lane 2: Western blot - Human FMR1 knockout U-87 MG cell line (Human FMR1 knockout U-87 MG cell line ab306664) at 20 µg

  Lane 3: Wild-type A549 at 20 µg

  Lane 4: Western blot - Human FMR1 knockout A549 cell line (Human FMR1 knockout A549 cell line ab288956) at 20 µg

  Secondary

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

  Performed under reducing conditions.

  Predicted band size: 71 kDa

  Observed band size: 75 kDa