This is normal. The data file (X and Y values) should be saved as a comma-delimited (.csv) file, and opened by clicking on the File menu in HEPB and selecting Open ( Fig. 5). The two columns of data are displayed in the memo field of the HEPB main interface for verification that the correct file has been opened. In addition, the name of the file is displayed at the bottom of the GUI, and remains there learn more until another file is opened. The user then clicks on the Analysis menu, and selects the Options submenu. This opens the Analysis Options window ( Fig. 6) where the user

can indicate to the program that the minimum and maximum values of the response variable in the data should be used as the fixed values of a and b, respectively (see Eq.  (1)), or alternatively, the user can provide the values for

the two constraints. The options for entering the values become visible upon choosing the “No” radio button. In a similar manner, the user can either accept the default options of iterating over the range of X values for estimating c and the range of − 50 to 50 for estimating d, or enter the desired range for either or both parameters. The user then chooses among five confidence levels for the prediction band (80%, this website 85%, 90%, 95% and 97.5%), which have been provided based on the algorithm by Shammas for the rapid approximation of the critical values of the Student’s t distribution (

Shammas, 2009). Finally, the user has the option of generating 500 values of the response variable within the observed range of the explanatory variable, based on the regression parameters estimated for the original data, by checking the Simulate data checkbox. After all the selections have been made (or default options accepted), the user then saves the options by pressing the Save Options button. While this button saves the options selected, it also alerts the user to any errors made on this page (e.g., invalid values) by means of messages at the bottom of the page (Fig. 7). After correcting all the errors, the user then presses the Save Options button again. This enables the Run submenu in the Analysis menu in the main HEPB form, which can now be selected. The analysis is then “Run.” 3-mercaptopyruvate sulfurtransferase The progress bar at the bottom of the HEPB main interface tracks the status of the analysis. The results (the estimated EC50 and Hill slope values for the regression, the cut-off values for the upper and lower limits of the prediction band, and the R2 value) are displayed in the memo field of the main form. These results are followed by the input values (X and Y), the expected Y values based on the Hill equation regression (Y-hat), the lower and upper limits of the prediction band for each X value at the confidence level chosen by the user, and the residual (Y–Ŷ, Fig. 8).