Dear all,
in our lab we are working on Outer Membrane Vesicles (OMVs). When we purify these vesicles we simply quantify the total protein content using nanodrop. Then we check the quality of the OMVs by running an SDS-PAGE normalized for 20ug total protein.
Curiously, we observe the following phenomenon every now and then: all samples with an absorbance index 260nm/280nm higher than 1.2 seem to contain something that is not protein, because on the gel there is less (stained) protein visible although samples are normalized.
We can exclude DNA as contaminant. So, what else could absorb at 280nm to cause this issue? Any ideas?
There a many reagents which also absorb at 280 nm. I do not know your procedure and the reagents used to isolate and purify the vesicles. Check all the used chemicals for possible absorption interference, for example detergents, RIPA buffer, imidazole ....
Peter
The ratio of absorbance at 260 nm and 280 nm is used to assess the purity of DNA and RNA.
A ratio of ~1.8 is generally accepted as “pure” for DNA; a ratio of ~2.0 is generally accepted as “pure” for RNA. If the ratio is appreciably lower in either case, it may indicate the presence of protein, phenol or other contaminants that absorb strongly at or near 280 nm. Good Luck.
http://www.nhm.ac.uk/content/dam/nhmwww/our-science/dpts-facilities-staff/Coreresearchlabs/nanodrop.pdf
https://tools.thermofisher.com/content/sfs/brochures/T123-NanoDrop-Lite-Interpretation-of-Nucleic-Acid-260-280-Ratios.pdf
Thank you, Badreddine. I know that the ratio is used for the purity of DNA. In contrast, a value near 0.5 also indicates purity of protein. Of course, it depends what one wants to know.
We are interested in the protein content of our OMVs. It is known that DNA occurs also in OMVs, but for some reason OMV sample with higher 260/280 ratios contain less protein while DNA content remains the same (we checked that!).
Thank also you, Peter. Our procedure is a standard protocol and thus contains always the same chemicals. The only differences are the actual vesicles that are composed of outer membrane and periplasmic compounds.
What Peter was asking you is to check the chemicals used in those protocols to see if there is something else absorbing at 260nm, like imidazole, maybe B-merpatoethanol, maybe even the fatty acids of the membranes
After ultracentrifugation we resuspend the pellet containing the OMVs in 1xPBS. In other words: water, salts and the OMVs themselves.
It looks from the comments and replies that likely a small molecule is responsible for the effect which is sometimes in the OMV sample (or its concentration is higher in some samples). I suspect it is a nucleotide analog like ATP or NAD/H. I do not think fatty acid or phopholipid can cause extra absorbance at 260 nm, unless chemically modified. What is the source of your OMV? Best: Karoly
Thank you, Karoly. Good point. We will consider this.
Anyway, the source of OMVs is E. coli BL21(DE3).
I was curious because many OMV is prepared from blood, which would open up other possibilities as well :-) E. coli is unlikely to contain "exotic" compounds, so I bet for nucleotide species. Hope you can figure out!
Best: Karoly
These ratios are meaningful when light scatter is not an added problem. With vesicles, light scatter is a problem. I hope that is not confounding your results. A variable result is due to a non-systematic error creeping in. Light scatter becomes profound as the wavelength decreases. The ratios further enhance errors.
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