[Protein-analysis] Re: Bradford Assay !

Eric say.no at spam.now
Fri Aug 4 04:35:03 EST 2006


anurag sharma wrote:
> 
> Dear All,
> I am new to this protein chemistry lab and I am trying to do this  Micro
> Bradford Assay. The problem is inspite of following protocols for making
> the reagent I am not able to get differential absorbance in blanks and
> samples. Can anyone suggest me the right protocol or point to the
> mistakes.
> Regards,
> Anurag Sharma
> Indian Institute of Technology, Delhi.

Here is a protocol that I have followed for 250µL microplates...

1. Premix the BioRad Bradford reagent with millipore (mp) H2O such that the
   final volume will equal a 1:5 dilution.

   Ex: 150µL mpH2O + 40µL Bradford reagent + 10µL sample

   The Bradford working solution (WS) should be at 21.1%, for 20mL use
   4.22mL Bradford reagent + 15.78mL mpH2O.

2. Filter the premix using Whatman #1 paper under gentle vacuum and store
   at 4-12°C.

3. Pipette the following volumes in the the microplate wells:

  Controls (#1-4):
   1µg/µL BSA standard 	0	2	4	6	8	10
   mpH2O		10	8	6	4	2	0
   Bradford WS		190	190	190	190	190	190

  Unknowns (#5-n):
   Sample		3	3	3	5	5	5
   mpH2O		7	7	7	5	5	5
   Bradford WS		190	190	190	190	190	190

 Note: This will give you 6 data points for each unknown, take the mean.
       You could also just use 3 replicates at 5µL.

4. Gently rock the plate for 4 min (or 5 min if your plate reader does not
   have a "shake" function).

5. Set your plate reader to shake the plate at varying frequencies for 1 min
   and then read the plate at 595 nm.


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