Hi everyone,
I have a problem about my bradford assay. I'm using bio rad bradford 1x dye reagent and when i am designed an assay with this reagent always something is wrong. i am following the procesure that protocol of reagent. I drawed a calibration curve with bsa standarts after that i am pippetted my samples with reagent the results are too much irrelevant each other. for example when my first reading is 0.13 with first sample, and second reading is 0.27 with same sample after 30 seconds later..so when i tried third reading with same sample and this time the result is 0.18..we tried almost everything filtered dye, changing sample, diluted different concentration standart etc...the most interesting thing is sometimes concentrate sample result is lower then the diluted sample result..the only thing is my reagents expiry date is pass.
i think the dye is not working because of passing its expiry date.
so here is my question, the reason of this situation is really just pass of reagent's expiry date? maybe different reason is exist but i do not decide that. Anyone experience like this situation?
It could be the expiration date, the thing is that you should not be able to construct a calibration curve either. I hate Bradford's assay, any variations in volume will give you different results. It is also a nonlinear analysis.
Hi Evren, I agree with Omar. Bradford assays are a pain in the gluteus maximus.
Throw it out and use the BCA assay from Pierce-Thermo Fisher.
I use the Bradford assay all the time without any problem. Here are some ideas as to what may be causing your trouble.
1. The reagent is too old and has become unstable, forming particulates. Check for this by eye.
2. Your cuvettes are not clean and the reagent is sticking to protein deposits on the sides. Clean your cuvettes with a specialized cuvette cleaning solution. Alternatively, if there is an absorbance plate reader available, conduct the microassay in disposable 96-well clear microplates.
3. You are not mixing your solutions well enough, or your are mixing them too forcefully and introducing bubbles.
4. You are not filling your cuvettes with enough solution, so that the meniscus is intersecting the light path.
5. Your protein is precipitating in the reagent. Some proteins do this.
6. Your samples contain an interfering substance. Detergents, in particular, are not compatible with the Bradford assay.
If you want to get sure whether the problem is due to the reagent expiry date or not, simply try to conduct your assay with a new reagent then if you still face a problem, so keep your eyes on the points that Prof/ Adam B Shapiro suggested.
Hi Evren
You can find some useful references on attachment 1
https://www.reddit.com/r/labrats/comments/53l4xx/bradford_assay_problems/
6
https://info.gbiosciences.com/blog/bid/164578/bradford-protein-assay-calculation-of-an-unknown-standard
8
https://www.sigmaaldrich.com/content/dam/sigma-aldrich/docs/Sigma/Bulletin/b6916bul.pdf
13
http://cst.ur.ac.rw/library/Food%20Science%20books/batch1/Food%20Science%20and%20Technology%20Published%20articles/Analysis%20Protocols/The%20bradford%20method%20for%20protei%20quantitation.pdf
14
http://home.sandiego.edu/~josephprovost/Biochem%20Protein%20Assay%2096%20well%20protocol%202014.pdf
16
https://www.montclair.edu/media/montclairedu/csam/nsftuesgrant/Module-6I-Protein-Concentration-Determination.pdf
17
https://tools.thermofisher.com/content/sfs/brochures/TR0057-Read-std-curves.pdf
18
http://wolfson.huji.ac.il/purification/PDF/Protein_Quantification/OZBioscienceBradford.pdf
19
https://tools.thermofisher.com/content/sfs/brochures/TR0057-Read-std-curves.pdf
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