I recently started using NanoVue to quantify total protein in saliva samples.
I have noticed that the values are always much lower than expected (about half). Does anyone have experience with this method?
What are the possible reasons for this to happen?
Thank you!
What method for protein quantitation are you using? Have you compared the Nanovue result to another method? Is it possible you have saturated the detection with too high a concentration? What are you using as the standard and blank?
Hi Nuno,
with nonovue check different mathods (molar extiction, wave lenght scan...). Some of them use a ratio between 280 and 260 value (es: molar extinction): check possible ligan of your protein (if your protein has a NAD+ bound you will underextimate your sample cause NAD+ absorbe at 260). Otherwise, for small protein check if few aromatic amino acids are present: absorption at 280 is provided by Trp (the major contribute), Tyr eand Phe.
Good luck..
Thank you for your answers.
I tested two methods for protein quantification, ProteinUV and Ab280.
I also tested Micro BCA (reading in NanoVue) which gave me inconsistent results.
I have read a series of dilutions of BSA made from a 2mg/mL comercial stock solution and the results were always lower than expected. For example, for a 1000 ug/mL concentration of BSA, the results were something like 650 ug/mL
I'm not familiar with the NanoVue. Does it have a variable setting for pathlength that might be set incorrectly.
When quantifying proteins by UV absorbance based on an expected extinction coefficient, you should denature the protein completely with 8M guanidine HCl to get the correct absorbance.
Determination of absolute protein concentration in saliva is difficult, because extinction coefficients of individual salivary proteins are not known. Moreover, saliva contains substantial amounts of proline-rich proteins, especially when collected under stimulating conditions. These do not absorb at 280 nm. You may give the method of Waddel a try: protein (mg/mL) = 0.144*(A215-A225). Dilute the protein sample so that A215 < 1.5.
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