I obtained a protein concentration from my sample 0.55 mg/ml, after I did filtration by using Amicon Ultra 0.5 centrifugal filter I measured the concentration again by Nanodrop and the result was 0.15 mg/ml, does this result make sense?
Don't use the Nanodrop for measuring protein concentration. In our experience, it is not reliable for this - it's great for RNA and DNA but not protein. Best way to measure protein concentration is using the BCA kit or similar.
Nanodrop is a valid instrument for determining protein concentration.
Typical problems could be air-bubbles, ergo make several measurements.
A significant drop of concentration as in your case, Eric, suggests loss of protein in the application of the centrifugal filter. The protein might interact with the filter or even pass it (hydrophobic protein? unfolded protein?).
Also, you need to consider what you are measuring against. Is it the same buffer as the proteins?
The NanoDrop is able to use BSA as a standard. I haven't tested it against a clear sample, but will in the near future.
What is the filter size? 10kDa / 30kDa? We are currently testing differences in recovery between these two.
- Badges
- Science method