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.
