I am trying to optimized a fluorescence based spectrometric assay by 96 well plate method which i was doing using cuvette. But the readings are not being consistent and acceptable. The plate I'm using is transparent one and not black plate. Is that the problem or something else ?
Hi, Tarun, which plate readre have you used? Have you tried any positive control? I mean fluoresent dyes whcih exited in the same wavelength as you samples?
There are a couple of reason why you dis not observe signal and we must know all deails to naswer your question..
But basically, first try the positive control.
What kind of plate reader do you have? If it's bottom read, then it has to be a clear plate of course, but, this can result in crosstalk and worse signal-to-noise. There are so-called ViewPlates with solid walls, but clear bottoms, you could try those.
If you have a top read option, I would recommend that, combined with a black plate. In my experience, this is best for fluorescence, eliminating cross talk and reducing background fluorescence.
I'm assuming of course that you're assay is identical to the cuvette one and that your plate is not just transparent but does not autofluoresce in the region you are interested in.
Thank you all for suggestions. The plate reader i am using is Biotek H4 multimode hybrid reader which has both top and bottom reading options. I tried both. For reducing the neighboring well interference, i kept wells empty around sample well, but it dint work. I tried different volumes like 100 ul, 200 ul, 300 ul. The 200 ul volume showed relatively less variation. I dont have any positive control dye although.
What exactly are your "symptoms", so to speak?
Reading-to-reading variation in the same well? or well-to-well variation of the same sample? How do your blanks/vehicle controls look?
Have you tried a spectral scan to confirm Ex/Em?
The H4 is a pretty badass system, so I have a hard time imagining it's the culprit.
well..the excitation wavelength is 380 nm and emission 460 nm. The problem is that the signal to noise ratio in sample wells is very close to that in blank (which has substrate in buffer but not sample)..the blank readings are in range of 8000 to 9000 units while sample ranges in 8500 to 10500. so the blank readings overlaps with some sample wells. There are well to well variations also.
For comparison, what does water or just buffer, no substrate read?
And you've used the same reagents--same lot--in a cuvette format and everything is fine?
I guess the two possibilities then are:
1) an issue is with your substrate. Maybe the substrate has oxidized or in another way degraded and is fluorescing in your blank? Without comparison to vehicle only and details of the assay/reaction it's hard to say.
2) an issue with your samples, they just are not reacting in the way you expect and there is no positive signal to really measure, hence no difference from the negatives.
Sometimes reagents just don't work right and need to be replaced.
I would try to come up with some positive control or standard curve if possible and re-check the reagents in the cuvette format that you had success with.
To check the substrate i used same experiment tubes with same proportion of buffer-sample-substrate in cuvette format and the blank (with substrate) comes down well below sample range..almost half the sample value
Well then I am stumped. All i can think is that the plates you are using simply have autofluorescence. Have you checked the signal from just water in the same plate? Do you have access to some other plate?
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