For identifying variations in protein folding, tryptophan fluorescence emission scan was performed using microplate reader (samples in black plate). The excitation wavelength was at 290 nm and emission spectral range between 320 and 400 nm. The peak did not start from the baseline. Can anyone suggest a change in method to overcome this problem and generate a graph with a sharp peak. It is not possible to bring the emission wavelength range down to 300 and 310 nm, since a minimum of 30 nm is needed as difference between emission and excitation wavelength.
Thank you for your response Peter. Our objective is to validate interactions via changes in the tryptophan fluorescence. We are not studying the exact patterns of changes in protein folding. I would personally feel that a microplate reader or an average spectrofluorimeter is good enough for that. Would you agree?
Thank you Nese and Peter for your valuable suggestions. I will let you know how it worked out!
Dear Subin, shift your excitation wavelength to shorter wavelength values, this will allow to let the emission wavelength range begin at shorter wavelength values. Tryptophan absorbance maximum in water is at 280nm, but you can also excite at 270 or 260nm without much intensity loss. Make sure to use µClear microplates or similar UV-compatible plate. Reducing bandwidth is always an option that allows to reduce the minimum excitation-emission distance.
Apart from this, I wouldn't call microplate readers "crap", as seen from another perspective the limitation of cuvette spectrometers to single samples could also be seen as "crap" feature, compared to measuring e.g. 384 samples within seconds... If fluorescent biological assays would exclusively be done in cuvettes, well, I don't know how many years of progress would be lost...
Of course cuvette spectrometer are more sensitive generally, and 90° detection is better, but tryptophan fluorescence is neither rocket science nor trace analysis...
Thank you Friedrich for your perspective on this, I have already completed the the assay in the microplate reader and have the data, we are curious as to how it will be in the cuvette based spectrofluorometerr! So, will be working on that soon!
Thank you all for your suggestions. I completed the assay using a spectrofluorometer. I got a better reading with the quartz based cuvette. Our hypothesis is that the plastic in black fluorescence plate might probably have been interfering with the readings earlier. I have attached a graph if you are interested in the observing the variation. Thank you all once again.
Hi Subin,
I have used both, plate readers and cuvette based instruments for fluorescence. I agree with Peter and Nesse - for experiments like the one you are doing, you need finer optics. No doubt the plate reader enables high-thoughput experiments which is a great advantage for certain kinds of experiments, but it is also limited due to its design for experiments like this one. Most importantly the spectra are going to be very broad on the plate reader, so it will be very difficult to see small shifts.
I highly doubt that your fluorescence readings in the pate reader were limited due to the plastic of the plate (although potential protein interaction with well plate surface is another issue). Plate readers are inherently poor at spectral deconvolution due to a large bandwidth and studying protein tryptophan does not make it any easier!
Good thing you have a spectrofluorometer! Good luck!
For the sake of microplate readers: many monochromator-based plate readers have variable bandwith, going down to as small as 2.5 nm. Which should be more than enough for analyzing tryptophan fluorescence. I used to tell the names of my company's products but not any more. They sacked me and a number of R&D colleagues for means of "cost optimization". This company is going down a steep road, where innovation does not belong to the goals any more... Plus, they facelifted the company website and screwed up all links... Dorks! Will take Google a while to update all search indexes.
Thats unfortunate. From my (admittedly quite limited) experience, the plate readers from different manufacturers either had good specs but terrible software, or good software but limited specs. (Not sure if this is even relevant here).
I wish you better pastures Friedrich.
The microplate reader I helped developing over the last four years has both, but I won't advertise it anymore...
The microplate reader not have a good resolution.
And I had a similiar problem, maybe your protein sample is not good at all, for example the protein could form amorphous aggregates that cause the light scattering and have bas specctra cause the scattering, I mean, the scattering overlap the signal of the Trp
Maybe you have to be more carefully with the protein to avoid the aggregation and meassure in a cubette not in a plate
Hi Subin R. C. K. Rajendran and Sheetal Tushir , have you resolved above mentioned spectrum issue.
Many black plates are fluorescent with UV excitation light. This interferes with tryptophan fluorescence spectroscopy.
I had good results with black Greiner 655209 plates.
- Badges
- Science topic
- Similar topics
- Physical Sciences
- Physical Phenomena
- Luminescence
- Fluorescence