BatchKi Reference Manual

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Plate Layout

The identity of enzymes, inhibitors, and substrates dispensed on each plate is given in the <Substance>...</Substance> section of the BatchKi data file. It contains of three subsections corresponding to the enzyme(s), substrate(s), and inhibitor(s) on each plate.

  <Substance>
    <Enzyme identical="[yes|no]"
            external="no">
...
    </Enzyme>
    <Substrate identical="[yes|no]"
               external="no">
...
    </Substrate>
    <Inhibitor identical="[yes|no]"
               external="no">
...
    </Inhibitor>
  </Substance>

There is no limitation on the number of different enzymes, substrates, or inhibitors, as long as the number of data points on each dose-response curve is at least four. From fewer data points, the apparent inhibition constant cannot be safely determined.

The identical attribute

This attribute can have one of two allowable values:

• identical="yes"
• identical="no"

If the identical attribute is set to `yes', the particular XML element (for the enzymes, the substrates, or the inhibitors) may contain only one identification symbol that will be applied to all wells on the given plate.

Example 1 - Identical Layout

    <Enzyme identical="yes">
HIV1PROT
    </Enzyme>
    <Substrate identical="yes">
BACHEM-1234
    </Substrate>

On the other hand, if the identical attribute is set to `no', the particular element must contain a tab-delimited table or spreadsheet with the number or rows and columns that is specified in the <Plate...> element above.

Example 2 - Non-identical Layout

<Plate rows="8" columns="3" id="...">
...
  <Substance>
...
    <Inhibitor identical="no">
IN11	IN12	IN13
IN21	IN22	IN23
IN31	IN32	IN33
IN41	IN42	IN43
IN51	IN52	IN53
IN61	IN62	IN63
IN71	IN72	IN73
IN81	IN82	IN83
    </Inhibitor>
...
  </Substance>
</Plate>

For compatibility with future versions of BatchKi, it is recommended to use rather short identification labels or codes for each enzyme, substrate, or inhibitor. Blank spaces and special characters (backslash `$\backslash$', colon `:', asterisk `*', etc.) are discouraged. It is best to think of the compound labels as file names on the given computer system. For example, on the Microsoft Windows system a file name cannot contain an asterisk.

The external attribute

The external attribute must have only one possible value:

• external = "no"

This rule also applies to all the other external attributes found in BatchKi data files. In fact, the external attribute can be safely omitted from the data file, because the program BatchKi assumes that all information is provided internally, within the BatchKi data file.

In contrast, the program PlateKi (also distributed by BioKin Ltd., see www.biokin.com/plateki) can work with primary data located externally on the hard disk. The external attribute is thus present only for compatibility purposes between the two companion programs, PlateKi and BatchKi.

Reserved inhibitor labels

The enzymes or substrates on each plate can have any arbitrary names or labels although, as was mentioned in the preceding paragraph, it is recommended that these labels do not contain blank spaces or special characters such as asterisk.

On the other hand, there are three special (or reserved) labels that can appear in the inhibitor subsection of the plate layout. They are the following case-sensitive labels:

• EMPTY
• CONTROL
• BACKGROUND

Unless the control parameter RequireNegativeControl is set to `no', every plate must contain at least one CONTROL well, which is defined as a well that contains no inhibitor but otherwise it contains the same amount (and kind) of enzyme and substrate as certain other wells on the plate.

The special label EMPTY is reserved for those wells that are to be excluded from the analysis, either because they truly do not contain any reactants, or because the enzyme assay in those wells somehow failed.

The label BACKGROUND is reserved for those experimental situations where a nonzero apparent reaction rate is observed even in the absence of enzyme catalyst. This situation might arise in monitoring the radioactivity of a captured phospho-peptides in protein kinase assays, where the reaction rate is approximated from a single-point measurement of radioactivity at the end of a fixed reaction period.

If a given plate contains any wells identified by the reserved label BACKGROUND, the rate equation used for the determination of inhibition constant is equation 4.3, where $v_b$ is computed as an average velocity from all wells identified by the BACKGROUND label. The background rate $v_b$ in equation 4.3 is considered as a fixed parameter.

On the other hand, if no wells on the given plate are identified by the BACKGROUND label, the rate equation used in the determination of inhibition constants is equation 4.6, unless certain special provisions are made for the handling of negative apparent reaction velocities (see section 4.5.11).

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Petr Kuzmic | Jul 12 2008