I know I might sound stupid. Currently I am performing binding assay to test the binding of the mustant domains for PDZ on E6 of the HPV 16, 18.
i prepared the Gst e6 and the four mutated pdz. However now I should do the binding assay. My previous test yield a different quantities of proteins however I need to have the same quantity from gst e6 to bind with the same quantity of pdzs.
could you please help me how to perform this quantification or a formula you work with?
thank you
I'll go through a worked example with just two hypothetical proteins
protein A = 53 kDa
Protein B = 61 kDa
(presuming you have purifed your proteins to >90%)
A = 0.00016 / 53000 = 3.018 nM
B = 0.00022 / 61000 = 3.606 nM
3. to ensure a 1:1 molar interaction in your binding assay you will need to dilute the more concentrated protein down to that of the least concentrated, in this case, protein B will need to be diluted to 3.018 nM so.....
61000 x 3.018 x 10 ^-9 = 0.000184 g/L = 184 ng/mL
4. Dilute protein B by the correct amount
184 / 220 = 0.836 = 8.36 uL of Protein B solution (220 ng/mL) = 1.66 uL buffer (or any equivalent proportion thereof!)
Hopo that helps!
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