Dear all,
I am performing BSA callibration curve with BSA but I am not getting nice callibrstion curve, just random points. I prepare concentration line by weighing BSA powder (from 10 microg/ml up to 2milig/ml) and dissolving in phosphate buffer pH 7.15. Then I mix BSA sample with Beadford reagent in plastic cuvette (500 ml BSA sample, 500 ml Bradford, 1ml total volume). I tried various BSA volume mixed with phosphate buffer(10, 20, 250 and 500microL and 500microL of Bradford), 10 min incubation in dark and then UV-Vis at 595 nm…I tried two Bradford reagents, so its not old…
Do you have any idea why I am not getting good callibration line?
thank you!
Dear Šárka Sovová
For measuring the concentration of total protein base on Bradford method, the following steps are carried down:
1. Preparing Bradford solution
2.Preparing BSA solution (in phosphate buffer pH=7.0 or Deionized Water) with different concentration (for example, 2-400 (μg mL-1)
3. Prepare standard curve by BSA solution
4. Add 400 μL of BSA solution to 1600μL Bradford Solution.
5. Incubate the samples at 30 0C for 30 min.
6. Measuring the absorption of protein solution at 595 nm.
7. Determining concentration of protein according to the obtained standard curve.
Bradford Solution:
Deionized Water, 85(mL)
Phosphoric Acid (85%), 10(mL)
Ethanol (96%) ,5(mL)
Cummasi Blue G250, 5(mg)
It is necessary to mention that in total range of 2-400 (μg mL-1), the relation of concentration and absorption is not linear. So, it must be divided to 3-4 ranges to prepare 3-4 standard curves. For example, (2, 4, 6, 8, and 10 μg mL-1), (20, 40, 60, 80, and 100 μg mL-1), (100, 200, 300, and 400 μg mL-1). Then, your final calculation must be done base on the absorbance of your main sample.
Mina Karimi-Avargani I prepared ranges 10-100 micro g/ml and 0.1-2 mg/ml and none of them gave me linear line...I will try your ranges and protocol to see if it works, thank you!
http://home.sandiego.edu/~josephprovost/Protein%20Assay%20Std%20Protocol.pdf,kindly follow this link.
https://info.gbiosciences.com/blog/bid/164578/bradford-protein-assay-calculation-of-an-unknown-standard?hs_amp=true,also check this link.
https://www.calstatela.edu/sites/default/files/dept/chem/07summer/201/bsa.pdf,also u can follow this link.
https://www.bio-rad.com/webroot/web/pdf/lsr/literature/4110065A.pdf,Also u can check this link,
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