Overview

  • Product name

    PTP1B Inhibitor Screening Assay Kit
  • Detection method

    Colorimetric
  • Sample type

    Purified protein, Inhibitor compounds
  • Assay type

    Enzyme activity
  • Product overview

    Abcam PTP1B Inhibitor Screening Assay Kit (ab139465) is a colorimetric, non-radioactive assay designed to measure the phosphatase activity of purified PTP1B. This 96-well assay is useful for screening inhibitors and modulators of PTP1B activity.

    The kit includes Human, recombinant PTP1B (residues 1-322; MW=37.4 kDa), expressed in E. coli as a positive control. The PTP1B inhibitor suramin is including as control for inhibitor detection.

    The detection of free-phosphate released is based on the classic Malachite green assay and offers the advantages of convenient, one-step detection and excellent sensitivity, without radioactivity.

     

  • Platform

    Microplate reader

Properties

  • Storage instructions

    Please refer to protocols.
  • Components 1 x 96 tests
    96-well Clear Microplate (1/2 Volume) 1 unit
    Phosphate Standard 1 x 0.5ml
    PTP1B Assay Buffer 1 x 20ml
    PTP1B Enzyme (Human, Recombinant) 1 x 5µg
    PTP1B Inhibitor 1 x 10mg
    PTP1B Substrate 1 x 1mg
    Red Assay Reagent 1 x 5ml
  • Research areas

  • Function

    Tyrosine-protein phosphatase which acts as a regulator of endoplasmic reticulum unfolded protein response. Mediates dephosphorylation of EIF2AK3/PERK; inactivating the protein kinase activity of EIF2AK3/PERK. May play an important role in CKII- and p60c-src-induced signal transduction cascades. May regulate the EFNA5-EPHA3 signaling pathway which modulates cell reorganization and cell-cell repulsion.
  • Sequence similarities

    Belongs to the protein-tyrosine phosphatase family. Non-receptor class 1 subfamily.
    Contains 1 tyrosine-protein phosphatase domain.
  • Post-translational
    modifications

    Oxidized on Cys-215; the Cys-SOH formed in response to redox signaling reacts with the alpha-amido of the following residue to form a sulfenamide cross-link, triggering a conformational change that inhibits substrate binding and activity. The active site can be restored by reduction.
    Ser-50 is the major site of phosphorylation as compared to Ser-242 and Ser-243. Activated by phosphorylation at Ser-50.
    S-nitrosylation of Cys-215 inactivates the enzyme activity.
    Sulfhydration at Cys-215 following endoplasmic reticulum stress inactivates the enzyme activity, promoting EIF2AK3/PERK activity.
  • Cellular localization

    Endoplasmic reticulum membrane. Interacts with EPHA3 at the cell membrane.
  • Information by UniProt
  • Alternative names

    • Protein-tyrosine phosphatase 1B
    • PTN1_HUMAN
    • PTP-1B
    • PTPN1
    • Tyrosine-protein phosphatase non-receptor type 1
    see all

Images

  • Dilutions of phosphate standard and a buffer blank were prepared. The 100 µL samples were mixed with 25 µL Red Assay Reagent and incubated at 30°C, 20 min to develop color. OD620nm was read on a microplate-reading spectrophotometer. Least-squares fit to the entire set of phosphate amounts, from 0 to 3 nmol.

  • Dilutions of phosphate standard and a buffer blank were prepared. The 100 µL samples were mixed with 25 µL Red Assay Reagent and incubated at 30°C, 20 min to develop color. OD620nm was read on a microplate-reading spectrophotometer. A more accurate correlation of OD620 to phosphate is obtained by separate fits of the data from 0 to 1 nmol and 1 to 3 nmol.

  • 2X PTP1B Substrate solutions (150, 50 and 20 µM) and 2X PTP1B Enzyme solutions (2 ng/well) were prepared and incubations at 30°C were performed. Reactions were then terminated by addition of 25 µL of Red Assay Reagent and OD620 read. OD620 readings were converted to nmol of PO4 with a phosphate standard curve. Each point represents the mean of two determinations.

  • Initial rates of IR5 (PTP1B Substrate) dephosphorylation by 2 ng of PTP1B were determined at 30°C and the indicated concentrations from 20 min. Time course plots (See Fig. 2 and Data Analysis). The line is a non-linear least squares fit to the Michaelis-Menten equation. The Km for IR5 (PTP1B Substrate) was 85 µM and the Vmax was 101 pmol/min.

  • Phosphate release from 75 µM PTP1B Substrate by 2.5 ng PTP1B (30°C) was measured at 10 min. Intervals for 30 min at suramin concentrations of 0 to 100 µM. Data were converted from OD620 to pmol of phosphate by means of standard curves (see Fig. 1B) and initial rates determined from best fits to the linear parts of plots of pmol phosphate vs. time (0-20 min. for 0 and 2 µM suramin, 0-30 min. for all other concentrations). The dose-response curve was derived from a least squares fit to the 4-parameter Hill-Slope model:

    (see Protocol Figure 4 legend text). The fitted parameter values were: top = 101.9 pmol/min; bottom = -3.5 pmol/min; IC50 = 9.5 µM; slope = 1.8.

  • PTP1B (2.5 ng) (red squares) or buffer (blue diamonds) was incubated for 20 minutes at 30°C with 75 µM PTP1B Substrate. Reactions were then terminated by addition of 25 µL of Red Assay Reagent and OD620 read. The Z’ factor for this assay was 0.84, (Z-factor = 1-((3SDpositive + 3SDnegative)/(mean positive – mean negative)). Dashed lines indicate the 3*Standard deviation range.

Protocols

References

This product has been referenced in:

  • Baburajeev CP  et al. Development of Novel Triazolo-Thiadiazoles from Heterogeneous "Green" Catalysis as Protein Tyrosine Phosphatase 1B Inhibitors. Sci Rep 5:14195 (2015). Read more (PubMed: 26388336) »
See 1 Publication for this product

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