Help - Fluorescence protocol for multiplate reader - (Aug/23/2010 )

Hi!

I'm looking for a protocol for some measurements of fluorescence of FMN (riboflavin mononucleotide).

I've got a multiplate reader (from P.E.) but i don't have any idea about some features of the protocol that I've need (for example: energy of the lamp, position of the counter, ecc.)

Somebody can help me?

Thanks in advance!

No protocol, but a few things to consider:

What wavelengths does the fluorophore excite and emit at?

What filters do you need? You will need an excitation filter to filter the incoming light into the correct wavelength to excite the fluorophore, and an emission filter to remove any other wavelengths that are produced by the fluorophore and are not needed for the experiment (i.e. not quantitative) and any light transmitted by the plate from the excitation.

The strength of the bulb shouldn't matter unless it doesn't produce light in the correct wavelengths to excite the fluorophore. Most plate readers have a mercury bulb or a halogen bulb.

The equipment manual should have instructions on how to get the plate reader to read the plate, it won't matter if it reads the whole plate, you'll just have to sort out empty wells from full ones in you use of the data.

I've already got some informations about wave lenghts of ex and em.
But for example i don't know how can i set for a good measurement the diameter of the lamp fissure and the counter position.
I keep on to looking for on the web for it!

Anyway thanks a lot!

Those things sound like the sort of thing that should be in the product manual... I would have a look for the instrument on the web and see if you can find a manual for it.