I was trying to do Bradford test to quantify total protein. Previously, i dont have any problem at all but recently when i carry out Bradgord test, my negative blank turn blue after adding Bradford reagent.
My protocol:
I put 10ul of water in microplate well, add in 10ul of diluted protein, then add 200ul of Bradford reagent. Leave at room temp 26C for 5 min and scan at 595nm.
For my negative blank, i just add in protein extraction buffer instead of protein sample. But it turn blue? is it because my reagent is old? My protein extraction buffer was prepared 2-3 months ago and i store at 4C, normally i would aliquot out the amount that i need then add in mercaptoethanol before use. For Bradford reagent, i prepare and filter using filter paper and store at 4C. what could have been the problem?
What kind of container did you use for the samples and blank?
Is there a chance that detergent made it into the blank?
@achim, i use 1.5ml microcemtrifige tube for sample, while for reagents i use amber glass bottle
As for the detergent, my extraction buffer contains SDS, phosphate buffer, edta, triton x-100 and mercaptoethanol
If I remember correctly, mercaptoethanol does also react with the reagent.
I would check each of the components you list individually with reagent.
In the list below you can easily find the compatible concentration or percentage of various substances with Bradford assay. You need to keep everything below the threshold in the test conditon.
https://zageno.com/l/ts-bradford-assay
I had the same problem long ago. The solution: use very clean or new glassware!
Yes, if you use glassware, make sure there are no residuals from the glassware dishwasher detergent. They definitely turn things blue!
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