Bradford confusion

[nawi]

Ok, I have seen that a lot of people have trouble with understanding this dilutions and concentration calculations.. and I would like to adress someone who has a lot of patience to explain to me this. I did Bradford the first time, so i had wide range of standards.. and i dont know how to calculate the concentration of standards!!! so if you explain me how to calculate this, i will know it once and for all,. i have my own theory but lets keep it a secret for now. so, to resolve this very "complicated issue", i will tell you that for the first standard i added
4 ul od BSA stock (1 mg/ml),
196 ul of buffer (PBS),
and 50 ul of Bradford reagent..
so i guess the TOTAL Volume is 250 ul? i dont even know that for sure, does the reagent volume counts also?

so that's pretty much it, and.. what is my concentration?

[protolder]

Hola, you have 4ug in 200ul<> 20ug/ml <> 20ng/ul, you have to relation this concentration to the OD 595. The volume of reagent is the same for all the samples and if you consider it, you have an error because the solution of BSA is the reference to evaluate unknown samples. Buena suerte