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Application Example: Biotinidase Enzyme Activity

Document ID: DQ283
Relevant to following products: WorkOut 2.0 Manta
Document description: Application Example: Biotinidase Enzyme Activity

In this assay, Biotinidase Enzyme Activity is calculated from absorbance measurements at 550nm. The microplate layout comprises a reagent blank well (A1), substrate blank well (A2), 5 standards and 29 unknowns in duplicate, each with their own blank group as follows:

The following analysis is required:

The substrate blank is corrected by the reagent blank
The standards and the blanks are corrected by the reagent blank
The unknowns are corrected by their own blank which has also been corrected by the CORRECTED substrate blank.
The activity of each unknown is measured from the standard curve.

The complication of the different blank corrections can be handled by breaking down the analysis into separate calculations/transforms:

Calculate the Net Substrate Blank, this is the Substrate Blank – Reagent Blank
Subtract the Reagent Blank from all the samples
Blank correct each Unknown well by its own blank group (the blank correction transform allows you to setup these associations)
Subtract the calculated Net Substrate Blank from the Blank corrected unknowns
Setup the standard curve fit using the standards (which have been corrected by the reagent blank) and calculate the concentrations of the unknowns which habe been corrected by their own blank and corrected substrate blank.

Example data files can be downloaded from here.

The Excel document shows this data processed in Excel.
The results file shows an example of this data.
The assay protocol has been setup to import data using the Excel Add-in. Setup the Excel Add-in as described below and select this assay protocol file to analyse the data.

Please note that the results file and protocol uses custom well types (substrate blank and reagent blank), this means that you also need to copy the attached WTYPES.DAT file to your WorkOut installation directory typically (c:\Program Files\Dazdaq\WorkOut 2.0) to open this results file. You may want to keep a back up of this file if you have already created custom well types of your own.

You can then launch the assay from raw data in Excel (from cells A1-L8). Alternatively, you can convert the protocol to one which measures data with the instrument as described below (offline to online).

Related articles:
DQ215: Can I transfer data from Excel to an Assay Protocol automatically?
How To: Transfer data from Excel to an Assay Protocol

DQ002: Is it possible to convert a protocol that imports data (offline) to a measurement protocol (online)?
How To: Convert an offline protocol to online…

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