What are the absolute readings for 260 and 280? It is hard to see that the ratio can change, unless there is some issue with the absolute readings. What I'm thinking is the possibility that your A280 reading is out of linear range as you concentrate the protein. If you dilute your concentrated protein before taking the readings, does your ratio return to ~0.63. Or is that what you are doing and now you have a new higher ratio?

Hey Nick Gee . So I took the 260/280 ratio around 1.5 mg/ml. found to be 0.63. Then I concentrated it to 3.8 mg/ml. then the ratio started getting weird. (to like 1 ). then I diluted and checked still it was showing higher reading.((260/280) - 1)

It’s hard to explain, but given that you now have two readings giving a ratio of 1, maybe your original ratio of 0.63 is suspect? Any chance you have nucleic acid contamination? What are your actual 260 and 280 absorbance readings?

My only guesses are

1) that maybe the desalting is insufficient and there is residual urea (or salt) which is leading to some absorbance in the UV, see here: https://webbook.nist.gov/cgi/cbook.cgi?ID=C57136&Mask=400

The ratio changing with concentration is perplexing but perhaps indicates insufficient refolding and there is some change in the stoichiometry of the bound urea with concentration,

2) (very unlikely) there is some unusual cofactor which binds / unbinds from your protein with changes in its concentration, leading to abs shifts. Maybe a coordinated metal which changes coordination state with overconcentration.

Recommendations - desalt again, better, more stringently, and preferably with a different method and compare then (e.g. dialysis, or UF/DF).

Prepare a dilution series of your concentrated protein and obtain UV/Vis specs, then compare these to the protein concentration using a colourimetrc assay to confirm the concentration by orthogonal method.

Hi Reshma,

I think Nick Gee is asking an important question. I'll add to it by saying, if you are using a standard UV-Vis spectrophotometer with a 1 cm pathlength cell, the concentrations you are talking about will be pushing the limits of the instrument at 1.5 mg/mL and beyond the capabilities of most instruments at 3.8 mg/mL

Using Beer's Law (A = Ɛbc) and assuming Ɛ280 = 1.55 mL*mg-1*cm-1 (yours will be different, but probably close to this), then the A280 will be:

A280 = 1.55*1*1.5 = 2.3

A280 = 1.55*1*3.8 = 5.9

An absorbance of 2.3 means only 0.4% of light is being transmitted. At 5.9, 0.0001% is being transmitted! That is very difficult to detect accurately. Spectrophotometers are starting to show linearity issues at 2.3, and only the very best instruments can handle an absorbance of 5.9. When the sample is too concentrated for your instrument, it will underrepresent your absorbance. Since your A280 is in the denominator, your ratio will increase.

So, to Nick's question, what are the absorbance values you are measuring? Best practice is to keep them between 0.1 to 1.5. Check your instrument to see what it recommends.

If you are using something like a Nanodrop, take lots of replicates to make sure you aren't looking at noise. For this instrument, you want higher concentrations since the path length, b, is very small. Check its recommendations. Let us know how it goes, and good luck!