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I have been measuring my protein solutions' concentrations by diluting them in water 20 fold with a final volume of 100 uL and then measuring the absorbances of these solutions in 96 well plates with plate reader. I don't remember having any problem up until today.

I used 20 mM phosphate buffer instead of water to dilute them today and measured the absorbances at 280 nm repeatedly three times and the absorbance for one of the solutions went up from 0.043 to 0.068 (absorbance of 20 mM phosphate buffer is 0.030 at 280 nM with same volume); I stopped measuring after third one but it would probably go higher as I measured until I hit a plateau.

I measured absorbances of two proteins and only one of them went up that much, other one went up from 0.071 to 0.088; if this were to be concentration dependent I would expect the second solution go even higher but it didn't happen.

I know there may be differences in UV absorbances if protein is folded or unfolded; would it be that dramatic? What is the reason for that increase? I will be grateful for an explanation and a practical solution to the problem.

NOTE: I increased the total volume to 200 uL by simply adding 100 uL of 20 mM phosphate buffer into all wells and the signal increase slowed down a lot; there is still some increase though with 0.001-0.003 increments in each measurement.

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Could it be a slow precipitation? –  Alan Boyd Feb 7 '13 at 21:03
    
I don't have any precipitation in stock solutions and I think it wouldn't precipitate after I dilute them. –  Engin Yapici Feb 7 '13 at 21:30
    
Is the stock solution in phosphate buffer or water? Depending on the protein, just changing buffers can induce folding changes and aggregation. –  MattDMo Feb 7 '13 at 21:45
    
It is in phosphate buffer in stock solution. –  Engin Yapici Feb 7 '13 at 22:20

2 Answers 2

up vote 2 down vote accepted

It looks like your protein concentrations are right on the limit of detection of the spectrophotometer, and changing the diluent buffer changed their concentrations. The samples may not have been thoroughly mixed after dilution and before measurement, so the varying measurements may simply be the solution coming to equilibrium. Temperature can also affect absorbance, so you should verify that your samples have equilibrated before drawing any conclusions. If the absorbance of your phosphate buffer is 0.03, I'd try to keep the sample absorbances above 0.075 or higher to avoid getting too close to the limit of detection. Also, make sure your buffer isn't too old or contaminated with something which could be affecting its absorbance characteristics.

I would suggest taking one or two protein samples and doing a dilution series (1:1, 1:5, 1:10, 1:20) in a large-ish volume (say 400 ul each, if you can spare it), vortex briefly to mix well, then measure triplicates of each dilution on your reader, along with appropriate blanks (buffer only). You will see differences between each measurement, but it should be quite small, depending on the accuracy and precision of your instrument.

Measured values will not be exactly the same from measurement to measurement, and it would take a lot more than three repetitions to determine if there was an actual drift trend occurring. Measure your sample plate every 5 minutes for an hour and plot the values (don't just eyeball them) to see if the machine may need to be serviced.

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These are really great suggestions but most of the time I don't have much protein. I tried using higher volumes but I don't get much absorbance; they are very close to background absorbance. If I use too much protein I both waste my samples and a huge meniscus forms which prevents me from determining the pathlength. I always pipette them very well to make sure they are well mixed too. But your suggestion for measuring for an hour and plotting a graph is a good idea, maybe UV lamp is too old. Thank you! –  Engin Yapici Feb 7 '13 at 22:25

Could be protein unfolding or changes in conformation. Absorbance at 280 nm is mainly due to the tryptophan residues, and can change substantially as these residues move from a more hydrophobic (buried inside the protein) to a more hydrophilic (exposed to the solvent) environment. Your protein may be reacting to the new buffer.

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According to the OP's comments the protein is being diluted into the same buffer (I think). –  Alan Boyd Feb 8 '13 at 15:56
    
The way I understood it, OP swtiched from water to phosphate buffer for this particular experiment, and is getting the result for one particular protein sample in that buffer. –  Drosophila Feb 8 '13 at 19:44
    
This article explains the effect of protein unfolding on amino acid absorbance with clarity detail: life.illinois.edu/biochem/455/Lab%20exercises/Photometry/… –  Drosophila Feb 8 '13 at 19:48
    
I diluted the protein with the same buffer I it was in. Sorry for the confusion. Thank you for the paper. –  Engin Yapici Feb 9 '13 at 1:15

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