0

I have 7mg of a 74kDa protein $A$ bound to a column. I want to bind another protein $B$ which is 19kDa onto protein $A$ so that every protein $A$ is bound (or as close to 100% binding as possible). I also know the $K_D$ of protein $A$ to protein $B$ is $9\cdot 10^{-8} M$, I am not sure if this is even relevant. How much of protein $B$ to I need to flow through to try to get complete binding?

One protein $A$ binds to one protein $B$. Can I just convert the 7mg into the amount that would result in one to one binding, i.e., 1.8mg of protein $B$? Then does the dissociation constant even matter? I don't have a ton of protein $B$ so I need to be frugal and calculate how much to use.

Ruochan Liu
  • 101
  • 1
  • I would strongly suggest starting with much smaller amounts (micrograms) and figuring out the conditions there before scaling up. Depending on what it is and how you're making it, 7 mg is a lot of protein. You don't want to waste it all (and however much Protein B you use) if you make a mistake. – MattDMo Nov 29 '22 at 22:29
  • @MattDMo The problem is the column is somewhat large, I may have to find a different way ultimately. However, I am still wondering about how to calculate the solution to this problem. – Ruochan Liu Nov 29 '22 at 22:30
  • Can you figure it out from https://en.wikipedia.org/wiki/Dissociation_constant ? If not, where are you stuck? – Bryan Krause Nov 29 '22 at 22:37
  • One other thing to consider - will Protein B bind to the column itself, either specifically or non-specifically? Also, what kind of column is it - bead-based like a typical liquid chromatography column, solid-phase like a GC column, something else? – MattDMo Nov 29 '22 at 22:37
  • @BryanKrause It seems from the dissociation constant, if I were to want all of it to be in the bound state, I would need an infinite amount of the protein according to the equation? – Ruochan Liu Nov 29 '22 at 22:50
  • @RuochanLiu That's correct; really, in biology and chemistry, complete anything is usually aspirational at best. Also as MattDMo points out, when you have other things to bind to the situation gets more complicated. So, you'll need to figure out what you really need based on what you're trying to do, which isn't particularly clear here so far. – Bryan Krause Nov 29 '22 at 22:58

0 Answers0