Anti-Alkaline Phosphatase antibody [EPR27506-72]

6 product images

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  Western blot - Anti-Alkaline Phosphatase antibody [EPR27506-72] (ab305305)

  Blocking and diluting buffer and concentration: Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS.
  
  The samples were run on a Bis-Tris gel. Performed under reducing conditions.
  
  False colour image of Western blot: Anti-Alkaline Phosphatase antibody [EPR27506-72] (ab305305) staining at 1/1000 dilution, 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, ab305305 was shown to bind specifically to Alkaline Phosphatase. A band was observed at 75 kDa in wild-type HAP1 cell lysates whereas no signal observed at this size in Alkaline Phosphatase knockout cell line. To generate this image, wild-type and Alkaline Phosphatase knockout HAP1 cell lysates were analyzed. First, samples were run on a SDS-PAGE gel then transferred onto an immobilon-FL PVDF membrane. Membranes were blocked in in Intercept® (TBS) Blocking Buffer diluted with an equal volume of 0.1% TBS 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 (IRDye® 800CW) preabsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution and Goat anti-Mouse IgG H&L (IRDye® 680RD) preabsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution.

  All lanes: Western blot - Anti-Alkaline Phosphatase antibody [EPR27506-72] (ab305305) at 1/1000 dilution

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

  Lane 2: Alkaline Phosphatase knockout HAP1 whole cell lysate at 20 µg

  Lane 3: Human adrenal gland at 20 µg

  Secondary

  Lanes 1 - 3: Western blot - Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Rabbit IgG H&L (IRDye® 800CW) preadsorbed ab216773) at 1/10000 dilution

  Lanes 1 - 3: Western blot - Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 680RD) preadsorbed ab216776) at 1/20000 dilution

  Performed under reducing conditions.

  Observed band size: 75 kDa

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  Western blot - Anti-ALPL antibody [EPR27506-72] (ab305305)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  
  ALPL is a glycoprotein of approximately 80 kDa and detected as a 55 kDa band after treated with Protein Deglycosylation MIX II.
  
  Exposure time: 37 seconds

  All lanes: Western blot - Anti-ALPL antibody [EPR27506-72] (ab305305) at 1/1000 dilution

  Lane 1: Untreated human adrenal gland tisuue lysate at 20 µg

  Lane 2: Human adrenal gland tissue lysate treated with Protein Deglycosylation Mix II at 20 µg

  Secondary

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

  Observed band size: 55 kDa, 80 kDa

  Exposure time: 37s

• 

  Western blot - Anti-ALPL antibody [EPR27506-72] (ab305305)

  Blocking and diluting buffer and concentration: 5% NFDM/TBST
  
  Low expression: A549 (PMID: 33858415 ).
  
  Exposure time: 
  
  Lane 1: 6 seconds
  Lanes 2 and 3: 180 seconds

  All lanes: Western blot - Anti-ALPL antibody [EPR27506-72] (ab305305) at 1/1000 dilution

  Lane 1: Saos-2 whole cell lysate at 20 µg

  Lane 2: HeLa whole cell lysate at 20 µg

  Lane 3: A549 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: 80 kDa

  Exposure time: 6s

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  Immunohistochemistry - Anti-Alkaline Phosphatase antibody [EPR27506-72] (ab305305)

  Immunohistochemical analysis of paraffin-embedded human serous ovarian tissue labeling Alkaline Phosphatase with ab305305 at 1/2000 (0.261 µg/ml) followed by ready to use Leica DS9800 (Bond® Polymer Refine Detection) kit.
  
  Positive staining on human serous ovarian carcinoma (PMID: 30979497).
  
  The section was incubated with ab305305 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 ready to use Leica DS9800 (Bond® Polymer Refine Detection) kit.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins.

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  Immunohistochemistry - Anti-Alkaline Phosphatase antibody [EPR27506-72] (ab305305)

  Immunohistochemical analysis of paraffin-embedded human spleen tissue labeling Alkaline Phosphatase with ab305305 at 1/2000 (0.261 µg/ml) followed by ready to use Leica DS9800 (Bond® Polymer Refine Detection) kit.
  
  Low expression in human spleen lymphocytes with positive staining on vascular endothelium.
  
  The section was incubated with ab305305 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 ready to use Leica DS9800 (Bond® Polymer Refine Detection) kit.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins.

• 

  Immunohistochemistry - Anti-Alkaline Phosphatase antibody [EPR27506-72] (ab305305)

  Immunohistochemical analysis of paraffin-embedded human adrenal gland tissue labeling Alkaline Phosphatase with ab305305 at 1/2000 (0.261 µg/ml) followed by ready to use Leica DS9800 (Bond® Polymer Refine Detection) kit.
  
  Positive staining on human adrenal gland (PMID: 31563571).
  
  The section was incubated with ab305305 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 ready to use Leica DS9800 (Bond® Polymer Refine Detection) kit.
  
  Heat mediated antigen retrieval was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution 2) for 20 mins.