Lidstrom: Molecular Devices Plate Reader

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Our System

• Machine = molecular devices: spectramax 190
• Software = Softmax Pro, version 5.3. (manual)

Order of reagents

Example 1:

• add 20-40uL cell extract per well. Add ~180 uL of mix of substrate & cofactors dissolved in buffer. Adding the large volume to the small volume should yield decent mixing.

Should I run a whole plate at once or rows/columns at a time?

• There are a few reasons you want to read the wells you load as quickly as possible. As soon as you mix all your reagents and cell extract, your reaction starts. If your assay has a non-linear reaction rate at short times (often the case) then you will lose some information at early timepoints. For example, often a reaction is fastest at small times, and slows down as the reaction proceeds and products build up.
• Unless you know your assay is highly linear for 30+ seconds, Janet recommends you run one cell extract with every substrate/no-substrate mix per scan of the machine, then move to the next one. OR, run all of your cell extracts with one substrate/no-substrate mix at a time.

Do I need to use the crystal plate or a plastic plate?

• NADH assays are run at 340 nm. This is right on the lower boundary for what wavelengths are suitable for our plastic so it shouldn't matter much. If the crystal plate is around, you might as well use it. At a minimum you will generate less trash.

Exporting Data

• If kinetic data needs to be exported and you can't get the data exported as one column per well:
  • Go to Settings --> Preferences
  • Change the settings to Time
  • Your files should look like this:

    

    best export format for Softmax Pro-kinetic data

    • Note: This assay ran for < 1 minute and Microsoft excel misinterpreted the times as minutes, not seconds. This may not be a problem if you run your assay longer, but it was a problem here.
  • (from manual)

Kinetic Data

• The machine can report Vmax in units of milli-Units/min or Units/second. What are "Units" you ask? It is just the slope. Multiply by 1,000 to get d(Absorbance)/min.

Plates

What type to use when detecting NAD/NADH

• You can chose between the crystal plate and disposable plastic plates. The decision is a trade-off between getting (possibly) better optics and less interferance at 340nm with a crystal plate, however the crystal plate builds up residue that may lead to inaccurate assay results.
• Disposable plate materials available: (source)
  • Methacrylate cuvettes are designed for accuracy throughout the VIS-UV spectral range from 285 nm to 750 nm.
  • Poly methacrylate cuvettes are ideal for concentration measurements in the 285 nm to 750 nm spectral range.
    • The Lidstrom lab doesn't stock these, as of 11/2013.
  • Polystyrene cuvettes are designed for assays throughout the 340 nm to 750 nm visible spectral range.
    • These are the default kind stocked by the lidstrom lab as of 11/2013. Janet got excellent results using them and they are only ~$1.50/plate.
  • UV cuvettes are ideally suited for measurements at 260 nm, 280 nm and in the visible range.

How to clean the crystal plate

From Amanda Smith 11/2013:

• https://groups.google.com/forum/#!topic/bionet.molbio.proteins/_ww-cMyYV2M
• http://www.researchgate.net/post/What_is_the_best_method_to_clean_a_quartz_cuvette
• Bio-Rad
  • We recommend the following cleaning procedure for quartz cuvettes:
    • Use diluted alkaline (0.1 M NaOH) or diluted acid (0.1 M HCl) solutions, followed by several washes with deionized water. You can also use a neutral pH detergent in warm water, followed by the 0.1 M HCl wash and deionized water rinses
    • Do not use hydrofluoric acid, as it can attack the quartz material
    • To avoid scratches, use only lens cleaning tissues or very fine cloth to wipe the surface of the cuvette window
    • Using an ultrasonic cleaner is not recommended, as it may cause the cuvette to break