Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4] (ab126820)

Overview

  • Product name

    Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4]
    See all Alkaline Phosphatase, Tissue Non-Specific primary antibodies
  • Description

    Mouse monoclonal [2F4] to Alkaline Phosphatase, Tissue Non-Specific
  • Host species

    Mouse
  • Tested applications

    Suitable for: WB, ELISA, IHC-P, Flow Cyt, ICC/IFmore details
  • Species reactivity

    Reacts with: Human
  • Immunogen

    Recombinant fragment corresponding to Human Alkaline Phosphatase, Tissue Non-Specific. expressed in E.coli

  • Positive control

    • Recombinant Human ALPL protein (AA: 18-502), HeLa and NTERA-2 cell lysates. This antibody gave a positive result when used in the following formaldehyde/methanol fixed cell lines: HepG2
  • General notes

    This product was changed from ascites to supernatant. Lot no’s high than GR307490-5 are from Tissue Culture Supernatant

Properties

Applications

Our Abpromise guarantee covers the use of ab126820 in the following tested applications.

The application notes include recommended starting dilutions; optimal dilutions/concentrations should be determined by the end user.

Application Abreviews Notes
WB 1/500 - 1/2000. Detects a band of approximately 57.3, 78.9 kDa (predicted molecular weight: 57, 78.9 kDa).
ELISA 1/10000.
IHC-P 1/200 - 1/1000.
Flow Cyt 1/200 - 1/400.

ab170190 - Mouse monoclonal IgG1, is suitable for use as an isotype control with this antibody.

ICC/IF 1/100.

Target

  • Function

    This isozyme may play a role in skeletal mineralization.
  • Involvement in disease

    Hypophosphatasia
    Hypophosphatasia childhood type
    Hypophosphatasia infantile type
  • Sequence similarities

    Belongs to the alkaline phosphatase family.
  • Post-translational
    modifications

    N-glycosylated.
  • Cellular localization

    Cell membrane.
  • Information by UniProt
  • Database links

  • Alternative names

    • AKP2 antibody
    • Alkaline phosphatase liver/bone/kidney antibody
    • Alkaline phosphatase liver/bone/kidney isozyme antibody
    • Alkaline phosphatase tissue nonspecific isozyme antibody
    • Alkaline phosphatase, tissue-nonspecific isozyme antibody
    • Alkaline phosphomonoesterase antibody
    • Alpl antibody
    • AP TNAP antibody
    • AP-TNAP antibody
    • APTNAP antibody
    • BAP antibody
    • FLJ40094 antibody
    • FLJ93059 antibody
    • Glycerophosphatase antibody
    • HOPS antibody
    • Liver/bone/kidney type alkaline phosphatase antibody
    • MGC161443 antibody
    • MGC167935 antibody
    • PHOA antibody
    • PPBT_HUMAN antibody
    • Tissue non specific alkaline phosphatase antibody
    • Tissue nonspecific ALP antibody
    • TNAP antibody
    • TNSALP antibody
    see all

Images

  • All lanes : Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4] (ab126820) at 1/500 dilution

    Lane 1 : Wild-type HAP1 whole cell lysate
    Lane 2 : ALPL knockout HAP1 whole cell lysate
    Lane 3 : Saos2 whole cell lysate

    Lysates/proteins at 20 µg per lane.

    Predicted band size: 57, 78.9 kDa



    Lanes 1 - 3: Merged signal (red and green). Green - ab126820 observed at 80 kDa. Red - loading control, ab181602, observed at 37 kDa.

    ab126820 was shown to specifically react with ALPL in wild-type HAP1 cells as signal was lost in ALPL knockout cells. Wild-type and ALPL knockout samples were subjected to SDS-PAGE. The membrane was blocked with 3% Milk. Ab126820 and ab181602 (Rabbit anti-GAPDH loading control) were incubated overnight at 4°C at 1/500 dilution and 1/20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preabsorbed ab216772 and Goat anti-Rabbit IgG H&L (IRDye® 680RD) preabsorbed ab216777 secondary antibodies at 1/20000 dilution for 1 hour at room temperature before imaging.

  • Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4] (ab126820) at 1/500 dilution + Recombinant Human protein ALPL (AA: 18-502)

    Predicted band size: 57, 78.9 kDa

  • ICC/IF image of ab126820 stained HepG2 cells. The cells were 4% formaldehyde fixed (10 min) and then incubated in 1%BSA / 10% normal goat serum / 0.3M glycine in 0.1% PBS-Tween for 1h to permeabilise the cells and block non-specific protein-protein interactions. The cells were then incubated with the antibody ab126820 at 1/100 dilution overnight at +4°C. The secondary antibody (green) was DyLight® 488 goat anti- mouse (ab96879) IgG (H+L) used at a 1/250 dilution for 1h. Alexa Fluor® 594 WGA was used to label plasma membranes (red) at a 1/200 dilution for 1h. DAPI was used to stain the cell nuclei (blue) at a concentration of 1.43µM. This antibody also gave a positive result in methanol fixed (100%, 5min) hepG2 cells used at 1/100 dilution.

  • All lanes : Anti-Alkaline Phosphatase, Tissue Non-Specific antibody [2F4] (ab126820) at 1/500 dilution

    Lane 1 : HeLa cell lysate
    Lane 2 : NTERA-2 cell lysate

    Predicted band size: 57, 78.9 kDa

  • Example of standard curves obtained using ab126820. Control antigen 100ng (black), antigen at 10ng (purple), antigen at 50ng (blue), antigen at 100ng (red)
  • Flow cytometric analysis of MCF-7 cells using ab126820 at 1/200 (green) and negative control (red).
  • ab126820, at 1/200, staining Alkaline Phosphatase, Tissue Non-Specific in paraffin embedded Human ovarian cancer tissues by Immunohistochemistry with DAB staining.

References

This product has been referenced in:

  • Parlato M  et al. Human ALPI deficiency causes inflammatory bowel disease and highlights a key mechanism of gut homeostasis. EMBO Mol Med 10:N/A (2018). Read more (PubMed: 29567797) »
See 1 Publication for this product

Customer reviews and Q&As

1-3 of 3 Abreviews or Q&A

Question
Answer



Below is the recommended flow cytometry protocol for use with ab126820:



Flow Cytometry Protocol

1. Solutions and Reagents

1.1. 1X PBS

1.2. Blocking buffer: 0.5% BSA in 1X PBS

1.3. Filter buffer: 0.1%Triton-X100 and Blocking buffer

1.4. The in ice cold 4% paraformaldehyde (1% solution - optional for storing samples)

1.5. Fluorescently-conjugated secondary antibody (various forms)



2. Protocol

2.1. Collect 1x106 cells/sample.

2.2. Wash cells once with blocking buffer.

2.3. Fix cells with 4% paraformaldehyde and incubate at 4oC for 30 min.

2.4. Wash cells once with blocking buffer.

2.5. Add 0.5 ml filter buffer and incubate at 0 oC for 15 min.

2.6. Wash cells twice with blocking buffer.

2.7. Incubate cells in blocking buffer for 10 min at room temperature.

2.8. Add primary antibody at the appropriate dilution (1/200 - 1/400 for ab126820) and incubate for 30 min at room temperature.

2.9. Wash twice with blocking buffer and incubate with fluorescently-conjugated secondary antibody for 30 min at room temperature.

2.10. Wash cells twice with blocking buffer.

2.11. Re-suspend cells in 1X PBS and analyze on flow cytometry. Samples can be kept in 1% paraformaldehyde at 4oC overnight.



3.1. Advice: Keep the cells in the dark on ice or at 4 oC in a fridge until your scheduled time for analysis. Analyze the cells on the flow cytometer as soon as possible.

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Answer

Thank you for your recent enquiries which have been forwarded to me as my colleague is away from the office today.

I wanted to contact you to let you know we have just received further information regarding the negative control. I can confirm this negative control was a no primary control.

I hope this will be helpful. If you have any further questions, please do not hesitate to contact us.

Read More

Answer

Thank you for contacting us in relation to the Anti-Alkaline Phosphatase (ab126820).

I am sorry for the delay in my reply. I can now confirm that the concentration of the antibody is 1 mg/mL. This value can therefore be used to calculate the appropriate dilution of your isotype control.

I hope this information has been of help. If you have any further questions, please do not hesitate to ask.

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