hi! excus me, I wanna asking about bradford method. I have made BSA as a standard protein, but after adding the bradford reagent to stadard (BSA + aquadest), it does not dissolve and becomes heterogeneous, as the suspension is formed. But, if you add the bradford reagent to the sample, it is dissolved and homogeneous. Does anyone know about that? What's wrong with that? I have tried three times, the results are the same.
Thank you for adding your answer.
Dear Ainur Rohmah Rohmah ,
The things that cross my mind are:
1. Is your concentration (range) correct? The stock solution should be in the range of a few mg/mL. Note: Only a narrow concentration of BSA is used (2-10 μg/mL) in order to create an accurate standard curve. See:
https://www.tau.ac.il/lifesci/departments/biochem/members/zor/pdfs/Zor_Anal_Biochem_1996.pdf
2. Is the batch of BSA you use still fresh? Since BSA should be stable, soluble and so on.
3. Consider using a buffer instead of water.
The following earlier discussion might give some answers, like adding the powder into the solvent instead of the other way around, try to avoid foaming and so on:
https://www.researchgate.net/post/How_do_you_dissolve_BSA_powder
Couple of remarks though: Do realize that this method (like every assay in this respect) is sensitive to the amino acid composition? So similar concentrations of different proteins will give different outcomes.
The use of BSA as standard protein for Bradford is more and more questioned it has been reported that BSA contains sites that binds hydrophobic substances which can give anomalies. Proteins like Ovalbumin or IgG are more recommended as standard protein for this assay.
See for good discussions and advices elsewhere here on Researchgate:
https://www.researchgate.net/post/Why_does_the_determination_of_the_protein_usually_use_bovine_serum_albuminas_a_standard
https://www.researchgate.net/post/Why_use_BSA_as_standard_in_bradford_assay
Good luck!
thank you all for answering my question. Related to the statement of Rob Keller points 2, I kept the BSA in the refrigerator but it could have been stored there for a long time ... would it affect the measurement? Is BSA not stable enough? thank you
Depends who else had his/her spatula in the bottle and how good you were at keeping moisture out. I usually kept my hygroscopic regents or proteins in a vacuum desiccator. And do not forget to change or regenerate the silica gel, when the indicator turns pinkish.
If you are talking about storing BSA SOLID at 2-8 degrees C, that shouldn't be a problem, but an aqueous solution, if not filter-sterilised, will be affected by microbial contamination over a period of weeks. It is very important to dissolve the BSA in a buffer solution: we use Phosphate Buffered Saline - to preserve the protein (albumin) in its 3D conformation. Always add BSA powder to buffer, when dissolving, to prevent 'clump' formation.
I hope this helps.
Good luck!
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