Anti-p75 NGF Receptor antibody [EP1039Y]

13 product images

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Immunohistochemical analysis of formalin fixed paraffin embedded human tonsil labelling p75 NGF Receptor with ab52987 at a concentration of 0.1µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

  Anti-p75 NGF Receptor antibody [EP1039Y] ab52987 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

  Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual).

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  Image from Abe SI. et al. PLoS One. 2017 Nov 30;12(11):e0188705. doi: 10.1371/journal.pone.0188705.

  Immunocytochemistry/ Immunofluorescence - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  The differentiation capacity of purified CD34⁺ cells cultured for 3 days in the presence or absence of ALK5i was evaluated by performing immunofluorescence analysis assessing whether CD34⁺ cells had changed to cells expressing p75 and/or α-SMA. Expressions of CD34, p75 and α-SMA were assessed by immunofluorescence on day 0 (F-H), day 3 in SP+f medium (I-K), or day 3 in the same medium as (I-K) but with ALK5i (L-N).
  

  Cultured re-aggregates were fixed in 4% PFA and embedded in paraffin. Sections (5 ∝m) were boiled in 0.01 M citrate (pH 6.0) with 0.1% Tween 20 for 10 min, washed three times in 0.1% Tween-20/PBS, transferred to blocking solution containing 5% BSA and 5% horse serum (Sigma) or goat serum (Invitrogen) in 0.1% Triton X-100/PBS for 1 hr, and incubated with primary antibody (p75 at 1/100 dilution) at 4°C overnight. After washing, the secondary antibody was added, and the sections were incubated for 2 hrs at room temperature. Microscopic images were obtained using a CCD camera (DP72, Olympus, Tokyo) mounted on a fluorescence microscope (BX60, or BX61VS-ASW, Olympus). Cultured cells on coverglasses were fixed in 4% PFA. Antigen retrieval was done by incubation with 100% methanol (-20°C) 10 min, and 0.3% Triton X-100 for 10 min.

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  Immunoprecipitation - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Lane 1 (input): PC-12 (rat adrenal gland pheochromocytoma) whole cell lysate 10μg
  Lane 2 (+): PC-12 whole cell lysate
  Lane 3 (-): Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab52987 in PC-12 whole cell lysate
  
  ab52987 immunoprecipitating p75 NGF receptor in PC-12 whole cell lysates. For western blotting, primary antibody used was ab52987 at 1.6 μg/ml. VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366), was used for detection at 1/1000 dilution. Capture antibody was used at 1:40 dilution (2μg in 0.35mg lysates).
  

  Blocking and diluting buffer: 5% NFDM/TBST.

  Exposure: 10 seconds

  All lanes: Immunoprecipitation - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Predicted band size: 45 kDa

  Observed band size: 75 kDa

  Exposure time: 10s

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Purified ab52987 staining p75 NGF receptor in paraffin embedded Human tonsil tissue sections by Immunohistochemistry. Antigen retrieval was by heat mediation using Antigen Retrieval Buffer (100X Tris-EDTA Buffer, pH 9.0) ab93684 (Tris/EDTA buffer, pH 9.0). Samples were incubated with primary antibody at 3.3μg/ml. A ready to use Goat Anti-Rabbit IgG H&L (HRP) was used as the secondary antibody. Hematoxylin was used as a counterstain. Positive staining on germinal centre of human tonsil.

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  Western blot - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Blocking and diluting buffer: 5% NFDM/TBST
  
  ab52987 fails to detect band of interest in Capan-1 (positive, PMID: 14613990) and brain lysates (positive, PMID: 21413144, 21541365), indicating its low affinity in some p75 NGF Receptor positive materials.

  All lanes: Western blot - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987) at 1/1000 dilution

  Lane 1: Capan-1 (Human pancreas adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 2: Mouse uterus tissue lysates at 20 µg

  Lane 3: Mouse brain tissue lysates at 20 µg

  Lane 4: Rat uterus tissue lysates at 20 µg

  Lane 5: Rat brain tissue lysates at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Predicted band size: 45 kDa

  Exposure time: 180s

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  Image from Li Y et al. Reprod Biol Endocrinol. 2011 Mar 8;9:30. Fig 2.; doi:10.1186/1477-7827-9-30; 8 March 2011 Reproductive Biology and Endocrinology 2011 9:30.

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Immunohistochemical analysis of murine uterus tissue with adenomyosis, staining p75 NGF Receptor with ab52987.
  
  
  Antigen retrieval was performed by heat mediation in citrate buffer (pH 6). Tissue was blocked with goat serum for 15 minutes before incubating with primary antibody (1/100) overnight at 4°C. A biotinylated goat anti-rabbit IgG was used as the secondary antibody and staining was detected using DAB.

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  Flow Cytometry (Intracellular) - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Intracellular Flow Cytometry analysis of PC-12 (rat adrenal gland pheochromocytoma) cells labeling p75 NGF Receptor with purified ab52987 at 1/80 dilution (1ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.
  

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  Immunocytochemistry/ Immunofluorescence - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Purified ab52987 staining p75 NGF receptor in PC-12 (rat adrenal gland pheochromocytoma) by ICC/IF (Immunocytochemistry/Immunofluorescence). Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% TritonX-100. Samples were incubated with primary antibody at 3.9 μg/ml. An AlexaFluor^®488 Goat anti-Rabbit was used as the secondary antibody at 2 μg/ml. DAPI was used as a nuclear counterstain. Confocal image showing cytoplasmic and Membranous staining in PC-12 cells.

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  Western blot - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Blocking and diluting buffer: 5% NFDM/TBST
  

  ab52987 fails to detect band of interest in hippocampus and cortex lysates (positive, PMID: 25180603, 28507518, 18930453, 20937383, 21059364), indicating its low affinity in some p75 NGF Receptor positive materials.
  
  Exposure: Lane 1-3: 8 seconds
  Lane 4-6: 180 seconds
  Lane 7-9: 30 seconds
  
  
  

  All lanes: Western blot - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987) at 1/1000 dilution

  Lane 1: SW480 (Human colorectal adenocarcinoma epithelial cell) whole cell lysates at 20 µg

  Lane 2: Human hippocampus tissue lysates at 20 µg

  Lane 3: Human brain cortex tissue lysates at 20 µg

  Lane 4: Neuro-2a (Mouse neuroblastoma neuroblast) whole cell lysates at 20 µg

  Lane 5: Mouse hippocampus tissue lysates at 20 µg

  Lane 6: Mouse cerebral cortex lysates at 20 µg

  Lane 7: PC-12 (Rat adrenal gland pheochromocytoma) whole cell lysates at 20 µg

  Lane 8: Rat hippocampus tissue lysates at 20 µg

  Lane 9: Rat brain cortex tissue lysates at 20 µg

  Secondary

  All lanes: Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

  Predicted band size: 45 kDa

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  Flow Cytometry (Intracellular) - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  Intracellular Flow Cytometry analysis of PC-12 (rat adrenal gland pheochromocytoma) cells labeling p75 NGF Receptor with unpurified ab52987 at 1/60 dilution (10ug/mL) (red). Cells were fixed with 4% paraformaldehyde and permeabilised with 90% methanol. A Goat anti rabbit IgG (Alexa Fluorr^®488) at 1/2000 dilution was used as the secondary antibody. Rabbit monoclonal IgG (Black) was used as the isotype control, cells without incubation with primary antibody and secondary antibody (Blue) was used as the unlabeled control.
  

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  Immunocytochemistry/ Immunofluorescence - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  ICC/IF image of ab52987 stained PC12 cells. The cells were 100% methanol fixed (5 min) and then incubated in 1% BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1 hour to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody (ab52987, 1 μg/mL) overnight at 4^oC. The secondary antibody (green) was Alexa Fluor^® 488 goat anti-rabbit IgG (H+L) used at a 1/1000 dilution for 1 hour. Alexa Fluo^® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1 hour. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.4 μM.
  
  

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  Western blot - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  All lanes: Western blot - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987) at 1/50000 dilution

  All lanes: PC12 cell lysate at 10 µg

  Secondary

  All lanes: goat anti-rabbit HRP labelled at 1/2000 dilution

  Predicted band size: 45 kDa

  Observed band size: 75 kDa

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  OI-RD Scanning - Anti-p75 NGF Receptor antibody [EP1039Y] (ab52987)

  We have systematically measured KD (the equilibrium dissociation constant between the antibody and its antigen), of more than 840 recombinant antibodies to assess not only their individual KD values but also to see the average affinity of antibody.
  Based on the comparison with published literature values for mouse monoclonal antibodies, Recombinant antibodies appear to be on average 1-2 order of magnitude higher affinity.