PTP1B Inhibitor Screening Assay Kit

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Functional Studies - PTP1B Inhibitor Screening Assay Kit (AB139465)

Inhibition of PTP1B by Suramin (PTP1B Inhibitor).

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.

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Functional Studies - PTP1B Inhibitor Screening Assay Kit (AB139465)

Dependence of PTP1B Kinetics on PTP1B Substrate (IR5) Concentration.

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.

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Functional Studies - PTP1B Inhibitor Screening Assay Kit (AB139465)

Z-Factor Analysis.

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.

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Functional Studies - PTP1B Inhibitor Screening Assay Kit (AB139465)

Phosphate Standard Curve.

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.

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Functional Studies - PTP1B Inhibitor Screening Assay Kit (AB139465)

Time Courses of PTP1B Phosphate Release from the PTP1B Substrate.

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.

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Functional Studies - PTP1B Inhibitor Screening Assay Kit (AB139465)

Phosphate Standard Curve.

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.