i have mixed a protein with certain concentration with a protein nano particles to loading on it. i separate the loaded particle by centrifuges.i want to measure the unloaded protein concentration in supernatant for determining the loading efficiently of my protein on nanoparticles indirectly. MW of my protein is 45kD and has a c terminal 6his tag. considering there are two types of protein in supernatant, how do i determine precisely my protein concentration?
Load known volume of a solution containing mixtures of proteins with different molecular size on a gel. Run SDS-PAGE gel with appropriate standard protein with known concentration. Then, you can estimate the concentration of each protein from the gel with the help of the standard protein. Use the Labimage software from biorad for determining the concentration of proteins from protein bands. It worked fine for me.
Dear Sara,
There are many methods to determine protein content. Alemu's approach, above, works well if you want to know the content of both proteins individually. If you are just looking for a measure of protein content before and after encapsulation, please review this link and select a method that fits your possibilities:
http://people.umass.edu/~mcclemen/581Proteins.html
Hope this helps, kind regards, Leo
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