Hello,
I am currently using LDH and bounding to toyo pearl chromatographic particles. I know I can determine the concentration of the protein in a buffer solution using the Bradford Assay Method. However, once I mix the solution with chromatographic particles, is it still a good test in order to determine the concentration of the protein in the slurry of the toyo pearl particles. If it is, how do you determine the concentration of the bound protein using this method?
You could measure the unbound protein concentration by spinning down the slurry and performing a Bradford assay on the supernatant. By comparing the concentrations of the original solution and the supernatant, you can calculate how much of the protein is bound.
I don't know these pearls particles but if you can achieve to separate the particles from the supernatant (by centrifugation for example) you can determine the protein concentration in the beginning and the protein concentration in the particle-free supernatant separately. The difference between both is the bound protein concentration (approximately). But this depends of course on the possibility of separation...
Thank you for your replies. Just one more question, how would you spin down the slurry and get to the supernatant? Is there a technique one can use?
You could use filtration to separate the bound toyopearl beads from the liquid phase, however do not filter to dryness, leave as a damp cake of resin on the filter. Filter of course using a buchner funnel and #54 Whatman paper will suffice.
A potential problem with filtration is that your protein may stick to the filter. You can test various kinds of filters to see which is least sticky. When you centrifuge a slurry of Toyopearl resin, such as in a microcentrifuge at 16,000 x g, does it not form pellet?
A lower speed centrifugation should work fine for a Toyopearl Resin so to not harm the bead itself.
If you measure the initial and final (supernatant) concentration by the fundamental volumetry law , you can calculate how much of the protein is bound.
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