Hello
I purified the elastin like polypeptides (ELPs) by inverse transition cycling (ITS). In each cycle of ITC I reduce the volume of PBS (to make it concentrated). In last cycle protein sample became highly viscous because of high concentration and I could not take it by pipette to measure the exact concentration by uv/vis spectrophotometer (after dilution).
Is there any way to measure the exact concentration of highly viscous protein sample? or how to take exact volume of protein to make it dilute for concentration measurement.
(I need concentrated sample for rheological measurements without freez drying method)
thanks
Hello! In such a case I would use weight instead of volume. However, it's desirable to know the exact density of your protein solution. But if dispersion around 15% is enough for you, you can use the density of water - for example, density of 40% whey protein is around 1.13 water densities at 20deg: http://www.ugr.es/~fcamacho/Originales/Trabajos%20Publicados/JFPE_2009.pdf)
You might have better success accurately pipetting such a solution very slowly with a gas-tight Hamilton syringe.
Very interesting question. We've done some work with highly viscous biological materials in a parallel plate rheometer and I agree it can be tricky. Assuming that your sample is not losing water through evaporation during the rheometry, perhaps you could do a destructive measurement after doing the mechanical characterization. This might include dry weight to estimate a protein weight fraction (as Azat mentions above) or possibly performing an chemical hydrolysis and measuring protein in a traditional way.
Best, John
www.akadeum.com
Is the final product "stringy" like DNA? Is it stable to warming? Adam is suggesting the use of what are called positive displacement pipettes and depending upon the volume size you need, there are different manufacturers with different orifice sizes and volumes. You might be able to get away with a slight warming of the sample without changing density?
How about quantitative determination of nitrogen by Kjeldahl method?
You can take the sample in calibrated tube so that you will know the volume of the sample and then you can dilute to read in UV spectrophotometer of you can use colorimetric method to estimate protein content.
Thank you all of you for your answers.
Actually I cannot heat my sample because of its aggregation at high temperature (ELP property)
The exact problem is to take sample by pipette tip as sample also stuck outside the tip and I cannot measure exact concentration.
- Badges
- Science topic
- Similar topics
- Biological Science
- Bioscience
- Biotechnology