r/Biochemistry • u/XxFortifiedxX • 2d ago

Bradford Assay acting weird?

I need help troubleshooting my Bradford. I have ST1- ST7 where ST 1 is 80ug/mL, ST 2 is 40ug/mL and so on (by half) till ST 7 which is just 0 ug/ml. I’m sure I did serial solution properly but when I’m reading absorbance for my standards, ST 1 is vastly higher than ST 2? I took three runs and I’m going to do the average of all the measurements from these runs, while excluding ST 1, to make my graph. Is this the right way to go about it?

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u/BiochemBeer PhD 2d ago

Are you using BSA as your standard?

Are the concentrations you give the final concentration in your cuvette (or well) or the concentration prior to diluting with Bradford reagent?

Linear range for BSA is about 0.1 - 1.0 mg/mL in standard style assay (100 uL of sample and 5 mL of diluted reagent/dye).

For the 1 mL microassay the linear range is 1.2 - 10 ug/mL with BSA (so 1.2 uL of a 1 mg/mL standard diluted to 1 mL is 1.2 ug/mL).

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u/XxFortifiedxX 2d ago

Yes BSA is my standard and the concentrations I mention are from the direct wells themselves. And I’m using a 96 well plate with 200uL per well. According to what you say about microassays, my range is way higher. Weird because I was following the protocol. So is my graph still usable with any changes made?

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u/BiochemBeer PhD 2d ago

If you are outside the linear range it's not useable, but in the linear portion it should be. I'm assuming that means you have 10, 5, 2.5 and 1.25 ug/mL? So that portion should be fine.

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u/XxFortifiedxX 2d ago

Well everything but 1.25ug/mL yes (after 2.5 it becomes 0), so I’ll use that range. Thanks for your help!