Hi,
this is a question to all experts on protein fluorescence:
Currently, I'm trying to measure the trp fluorescence of a protein in buffer.
It shows a nice signal, but unfortunately, results are really fluctuating, even for the repeats of one measurement.
I also observe something like bleaching of the protein fluorescence after a short time.
Can you explain these fluctuations (quenching by lysine side chains or oxygen??) and do you have any ideas to stabilize the system?
Looking forward to your answers =)
BW, Ina
Is it a problem related to the instrument or sample?
Is your sample transparent or are there any floating particles in your sample?
The instrument should be ok, all other applications work well.
Protein solution is transparent.
Ensure that you are recording your fluorescence spectra in S/R mode. This will take care of variations in lamp intensity. The variations which you are seeing may be due to this.
In your time scan expts, recording for very long duration (160 mins, figure 1) results in decrease in fluorescence intensity due to photobleaching.
ok thanks, do you have an idea how this photobleaching works in my case?
A common problem with proteins in cuvettes is that the proteins may contain cationic patches or even have cationic net charge, so that relatively rapidly they will adsorb on the cuvette walls. This is a particular problem with protein since they are often present at very low concentrations, often in the low µg/l range and even less than 1 µg/l. It will typically leads to slowly decreasing fluorescence after addition of your protein stock in the cuvette. In my experience, with typical modern fluorometers with blinking lamps (i.e. it is only on when measuring data points) and with typical protein concentrations of recombinant proteins this is usually far more significant problem than photobleaching. If you have 450 W Xenon lamp blazing on the sample continuously, or if you have milligrams/liter concentration proteins, then it is a different matter.
A very simple way to check for this is that you take two similar cuvettes, add the buffer. Add your protein into one of the cuvettes. Keep it in dark for half an hour. Measure fluorescence for 30 min or so. Then add protein into the other cuvette, and immediately start the measurement for 30 min. If it is photobleaching, then they will both start from the same value, let us say 250 a.u., and decrease similarly over time. If it is adsorption, then your cuvette that waited 30 min in the dark will start from the values that the cuvette you started measuring immediately ends at.
But if you take a look to the first diagram I attached here, I think this argues for a radiation dependent process (comparison of open and closed shutter)?
Oops, sorry, I had not noticed that. It seems that you have very high protein concentration (50 µg/ml or 50000 µg/l), so the issue I discussed is not very relevant. Clearly it seems that protein aggregation or anything like it is not responsible, since it does not happen with the one that has been kept in dark. I suppose that your fluorometer does not use continuous illumination either, or you would notice that with any protein. SNR appears to be fairly good, so you could decrease the bandwidth to decrease illumination. Just to exclude any instrumental cause (we once had problem with a monochromator that stuck in non-nominal positions randomly, causing weird behaviour), you could use just bovine serum albumin and repeat the experiment to see that it does not happen similarly to your protein of interest in the same buffer. If it does not, then I would look into the tertiary structure and sequence of the protein.
There are a number of issues that could be happening. First, Tryptophan is notorious for photobleaching. Try reducing the excitation shutter to see if you can get a stable intensity. Second, Tryptophans are often times near cystine bonds which can quench or even be disrupted upon tryptophan emission which can cause odd intensity fluctuations (I saw this once in one of my mutants). Best suggestions I have are to decrease the excitation shutter or put an ND filter in front of your sample, try to purge the sample of O2, use an bandpass filter after your excitation light to remove lower wavelength excitation light, and possibly try exciting your sample with 295 or 300 nm excitation light to remove excitation of tyrosine residues. Cheers
Dear researcher,
I sent you a paper about tryptophan fluorescence of alpha-Crystallin (from ResearchGate).
This is may be interesting for you.
Hello, sorry to reopen this old thread. Juha-Matti Alakoskela , could you elaborate on the adsorption problem you mentioned? I seem to have exactly that issue with my experiments - are there any suggestions how to reduce the adsorption to the cuvette walls, like surfactants or similar? Thanks a lot!
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