How do I go about making a set of protein standards for protein quantification v - (Jun/02/2014 )
I have been given a protein of unknown concentration and have been asked to make a set of standards of a protein with a known concentration so we can quantify roughly via dot blot.The aim is to use a floursescent secondary antibody and to visualise via odessy.
I need to know how to work out the calculation part. I would like to learn both mass and molar.
For example, the information I have for my standard protein is 30mg/ml and 550uM. I want to put for example 1ul dots on the membrane for my blot. I might want several dilutions of my standard, for example ranging from 20pg to 100ng. How do I work it out to get varied concentrations from the stock to my 1ul volumes on my blot?
Also, should I do varied dilutions of the unknown protein?
Thanks, I'm so new to theis and very confused
30mg/ml or 30ug/ul means you have 30ug in 1ul. Now if you make dilutions like 1:100 and 1:1000, you will get 300 ng and 30 ng respectively. Same way you can dilute it further till you reach to 30pg/ul. Accordingly you can add different dilutions to make standards with keeping volume constant.
Hey I get it, thank you so much.