Anti-SIK3 (phospho T469) antibody [EPR20877-104]

6 product images

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  Western blot - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633)

  Blocking and diluting buffer and concentration: 5% NFDM/TBSTThis blot was developed using a higher sensitivity ECL substrate.
  

  Exposure time: 114 seconds

  All lanes: Western blot - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633) at 1/5000 dilution

  Lane 1: Mouse hippocampus tissue lysate (Untreated membrane) at 10 µg

  Lane 2: Mouse hippocampus tissue lysate (Phosphatase treated membrane) at 10 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

  Predicted band size: 140 kDa

  Observed band size: 70 kDa

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  Western blot - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633)

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

  Data was kindly provided by Dr. Qinghua Liu, NIBS.

  The 1st Ab working concentration was 5 ug/ml.

  All lanes: Western blot - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633) at 0.522 mg/mL

  All lanes: Mouse cortex tissue lysate at 4 µg

  Predicted band size: 140 kDa

  Observed band size: 150 kDa, 70 kDa

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633)

  Immunohistochemical analysis of paraffin-embedded Mouse cerebrum tissue labelling SIK3 (phospho T469) with ab225633 at 1/1000 (0.522 ug/ml) dilution followed by a ready to use Rabbit specific IHC polymer detection kit HRP/DAB (Rabbit specific IHC polymer detection kit HRP/DAB ab209101). Cytoplasmic staining on mouse cerebral cortex without alkaline phosphatase treatment (image A). No signal was detected when tissues were treated with alkaline phosphatase (image B). The section was incubated with ab225633 for 30 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 (Rabbit specific IHC polymer detection kit HRP/DAB ab209101).

  Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

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  Immunocytochemistry/ Immunofluorescence - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633)

  Immunocytochemical analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized 293T cells labelling SIK3 (phospho T469) with ab225633 at 1/100 (5.22 ug/mL) dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) antibody (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 (2 ug/mL) dilution (Green). Confocal image showing cytoplasmic staining in 293T cells transfected with flag-tagged SIK3 WT expression vector, and no staining in 293T cells transfected with flag-tagged SIK3 T469A expression vector. Anti-FLAG mouse monoclonal antibody was used to counterstain tubulin at 1/500 (2 ug/mL) dilution (Red). The Nuclear counterstain was DAPI (Blue).
  

  Secondary antibody only control: Secondary antibody is Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/1000 (2 ug/mL) dilution.

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  Flow Cytometry (Intracellular) - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633)

  Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized 293T (Human embryonic kidney epithelial cell) cells transfected with flag-tagged SIK3 WT expression vector cells labelling SIK3 (phospho T469) with ab225633 at 1/500 dilution (0.1ug) (Right) compared with a Rabbit monoclonal IgG isotype control (Rabbit IgG, monoclonal [EPR25A] - Isotype Control ab172730) (Left). A Goat anti rabbit IgG (Alexa Fluor® 488) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution was used as the secondary antibody.

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  Dot Blot - Anti-SIK3 (phospho T469) antibody [EPR20877-104] (ab225633)

  Dot blot analysis of SIK3 (phospho T469) using ab225633 at 1/1000 (0.522 ug/mL) dilution followed by a Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (Goat Anti-Rabbit IgG H&L (HRP) ab97051) at 1/100000 dilution.
  

  Lane 1: SIK3 (phospho T469) peptide

  Lane 2: SIK3 non-phospho peptide

  Exposure time: 3 minutes.

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