protein quantification by bradford - (May/30/2009 )
Hi all,
I am going to measure the amount of protein in cell lysate and I am going to use Bradford methed. I prepare Bradford solution by dissolving 10mg coomassie blu in 5ml ethanol and then 10ml phosphoric acid and at last make the volume to 100ml by dH2O. the solution turns blue after adding water but it says in protocols that it should be brown red. I filtered it several times but there isn't any sign of red or brown color. even with 0.2 filter it turns completely colorless. can I still use the blue solution or should I do a trick or missing step to have the desired color?!
Hi,
I generally use the commercially available Bradford and it is brown. It turns blue in the presence of protein or detergents. So maybe its something in yr glassware or reagents which is making the bradford turn blue.
However, I use bradford only for rough estimation. If you want accuracy then its better to go for BCA or lowry.
Best,
TC
mtrnbh on May 30 2009, 12:19 PM said:
I am going to measure the amount of protein in cell lysate and I am going to use Bradford methed. I prepare Bradford solution by dissolving 10mg coomassie blu in 5ml ethanol and then 10ml phosphoric acid and at last make the volume to 100ml by dH2O. the solution turns blue after adding water but it says in protocols that it should be brown red. I filtered it several times but there isn't any sign of red or brown color. even with 0.2 filter it turns completely colorless. can I still use the blue solution or should I do a trick or missing step to have the desired color?!
Although the commercially available reagent (Bio-Rad) is easiest, home-made Bradford reagent is dirt-cheap and works very well indeed.
Brilliant blue (which was developed as a wool dye) comes in two "shades" G-250 (greenish) and R-250. Did you make the "standard" mistake and use the R-250 version? Bradford only works when the G-250 version is used.
Otherwise your recipe is fine.
Hope this helps
thank you for your reply. you are right that the dye may be R250 instead of G250 because I took it from someone in a microtube and didn't see its vial; although she said its G250 she may have been wrong!
thanx