i am new in the field of protein isolation and quantification. i have to do Bradford's protein assay i got G- 250 coomess brillient blue dye form sigma but i don't know how to make its solution for testing my sample and making standard cure of BSA. i just wana know about protocol of G250 protein binding dye solution.
cheers
Aslam
Hi,
First of all, I really do not recommend you to use homemade Bradford reagent (it is unstable, and gives unreliable results, and is not worth a few cents of savings). You would be better off with one of the ready-to-use products, be it from Sigma, Bio-rad, Pierce or whatever. They always supply a protocol with their product.
If you really want to prepare the solution yourself, this is the original paper: Bradford, M. M. (1976) A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of
Protein Utilizing the Principle of Protein-Dye Binding. Anal. Biochem. 72:248-254
The recipe is as follows:
Home made reagent:
Dissolve 100 mg of Coomassie blue G250 in 50 mL of 95% ethanol. Mix with 100 ml of 85% phosphoric acid and made up to 1 L with distilled water. Filter through Whatman No. 1 filter paper and then store in an amber bottle at room temperature. It is stable for several weeks. Filter before use if there is any dye precipitation.
Finally, remember that Bradford is not compatible with detergents in your protein buffer. If you have any, you might want to consider BCA method or automated stuff like Bioanalyzer or Experion.
Cheers and good luck!
Kris
Dear Aslam Muhammad , Bradford is a relatively sensitive method in protein assay. I utilized this method to quantification of many proteins. In this method you can use micro-bradford assay that is more sensitive. In the first you should prepare standard protein comprising BSA 1000ug/ml , 100ug/ml and 10um/ml then you should added 200 ul of Bradfor solusion in the each wells of 96 well plate and then 10ul of each standard should be added to each wells and simultaneusly test samples too (duplicate or triplicate) . Less than 3-5 min the OD of samples should be detected with ELISA reader in 595 , 600 0r 610 nm. Now you have 3 OD and three known concentration and you can design a gragh the gives a formula. then the OD of tests sample can be used in the formula and the concentration of the test sample calculated as ug/ml
if you know the extinction coefficient of your protein at some wavelength close to 280, you can calculate it using beer-lambert's law. however, your protein must be pure in order to do so or else other proteins will contribute to your absorbance reading.
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