I am trying to study protein aggregation based on light scattering effects using UV - Vis spectrophotometer. I took a spectrum measurement from 300 nm to 500 nm but I am getting improper values at longer wavelengths (beyond 400 nm). What wavelengths are used to analyze protein aggregation? Does the wavelength of light used to scatter depend upon the protein size? Can anyone please share some information relevant to this work.
To study the scattering with an absorption spectrometer is not the most direct way. But the scattering of the light will contribute to the spectrum on an absorption spectrometer since it removes some of the incident light from the detector. If you know the size of the detector, if you use a fixed slit width, you may do some predictions. Rayleigh predict a scattering efficiency that depends on 1/(wavelength)^4 . Mie theory predicts the exact form for the scattering of spherical objects depending on their size. On a practical point of view, scattering on a absorption spectrum, will appear as a flat offset from 800nm down to 500nm that will rise in the UV. For a qualitative detection of scattering and aggregation, it is perfect. For a quantitative study you had better to use an angle resolved scattering instrument or a dynamic light scattering instrument.
- Yes, you have to do DLS for study of protein aggregation.......UV-Vis absorbance is independent of protein aggregation......in my all case of study.
Thank you all for your replies. I will be doing DLS but I also wanted to make sure that I cannot make direct predictions from my absorbance data.
I work in the same topic. The size exclusion chromatography with a refractive index detector could be the best way to observe the aggregation of proteins. In UV/VIS the aggregation (Monomer, Dimer, Trimer) have the same absorbace.
Hello Anitha,
you can chose any wavelength in the range starting from 350 -650 nm but you have to have confirm yourself that either your protein of interest or any ligands which can not be absorbed on selected wavelength. You can simply noted down the aborbance at selected walvelength (simple read method) and not need to do scaning of spectra in selected range.
You can use a spectrophotometer to observe insoluble protein aggregation - you can even use a nanospectrophotometer for that (like the Nanodrop). You won't be looking at UV absorption though - you'd be looking at turbidity (the "cloudiness" of your solution - scattering of light from fine protein aggregates suspended in your solution). For that I find 340 nm works best. Of course you need to run some controls - positive (for example your protein sample with a precipitating agent like ethanol), and negative (your protein sample in a strong detergent, like 8M urea).
Good luck!
- Badges
- Science method
- Similar topics
- Methodology
- Absorption Spectroscopy