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I've recently started doing Bradford Assays for my samples and my standard curve has been non-linear and I have been getting low R values (.90-.95). I initially thought the error was in pipetting, most likely in adding BSA while establishing the standards but I have repeated in care and somehow still get low R values. Are there any other possible sources of error?

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  • You could be measuring outside the dynamic range of the assay (you can only add so much / so little BSA to a certain amount of reagent).
  • You could be outside the linear range of your spectrophotometer, usually under 1 is fine, although some old/crappy ones might not even be reliable over 0.5.
  • If you're fast you're measuring your different concentrations also at different times. Bradford is a little bit time-dependent, so waiting the recommended time before measuring your standards is not a bad idea.

Probably it's still pipetting though, try pipetting at least 20 uL in every dilution step and see if you're still off.

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