Anti-SIGLEC8 antibody [EPR24419-56]

4 product images

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  Flow Cytometry (Intracellular) - Anti-SIGLEC8 antibody [EPR24419-56] (ab305297)

  Flow cytometric analysis of 2% paraformaldehyde fixed 0.1% Tween-20 permeabilized HEK-293T (human embryonic kidney) cells transfected with a human SIGLEC8 expression vector containing a myc-his tag cells labelling SIGLEC8 with ab305297 at 1/50 dilution (1ug)/ Right compared with a Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) / Left isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081) at 1/2000 dilution was used as the secondary antibody. Live cells were surface stained with ab305297, then fixed with 2% PFA followed by intracellular staining with Myc-tag conjugated to Alexa Fluor® 647.

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  Immunocytochemistry/ Immunofluorescence - Anti-SIGLEC8 antibody [EPR24419-56] (ab305297)

  Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized HEK-293T (human embryonic kidney epithelial cell) cells labelling SIGLEC8 with ab305297 at 1/1000 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (Green). Confocal image showing cytoplasmic staining in HEK-293T cells transfected with a human SIGLEC8 expression vector containing a myc tag. The image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

  Myc-Tag Mouse mAb (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 dilution (Red).

  The Nuclear counterstain was DAPI (Blue).

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution.

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  Immunoprecipitation - Anti-SIGLEC8 antibody [EPR24419-56] (ab305297)

  SIGLEC8 was immunoprecipitated from 0.35 mg HEK-293T (human embryonic kidney epithelial cell) cells transfected with a human SIGLEC8 expression vector containing a myc-His-tag®, whole cell lysate, 2 μg with ab305297 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab305297 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(VeriBlot for IP Detection Reagent (HRP) ab131366) was used at 1/5000 dilution.
  
  Lane 1: HEK-293T (human embryonic kidney epithelial cell) cells transfected with a human SIGLEC8 expression vector containing a myc-His-tag®, whole cell lysate, 2 μg
  
  Lane 2: ab305297 IP in HEK-293T cells transfected with a human SIGLEC8 expression vector containing a myc-His-tag®, whole cell lysate
  
  Lane 3: Rabbit monoclonal IgG (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) instead of ab305297 in HEK-293T cells transfected with a human SIGLEC8 expression vector containing a myc-His-tag®, whole cell lysate
  
  Blocking and dilution buffer and concentration: 5% NFDM/TBST.
  
  Exposure time: 3 seconds

  All lanes: Immunoprecipitation - Anti-SIGLEC8 antibody [EPR24419-56] (ab305297) at 1/30 dilution

  All lanes: HEK-293T (human embryonic kidney epithelial cell)

  Secondary

  All lanes: Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (VeriBlot for IP Detection Reagent (HRP) ab131366) at 1/5000 dilution

  Observed band size: 60 kDa

  Exposure time: 3s

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  Western blot - Anti-SIGLEC8 antibody [EPR24419-56] (ab305297)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  Exposure time: 1 second

  All lanes: Western blot - Anti-SIGLEC8 antibody [EPR24419-56] (ab305297) at 1/1000 dilution

  Lane 1: HEK-293T cells transfected with a human SIGLEC8 expression vector containing a myc-His-tag®, whole cell lysate at 20 µg

  Lane 2: HEK-293T transfected with an empty vector (vector control), containing a myc-His-tag®, whole cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat Anti-Rabbit IgG H&L (HRP) (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution

  Observed band size: 60 kDa

  Exposure time: 1s