Bradford Assay !

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 formaking 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. *

anurag sharma wrote:

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.

Why not try the easy to use Quant-it kits from invitrogen which uses a
fluorescent dye and can be used in a microplate as well as a cuvette
format (Q33210). Even easier use the cheap new Qubit fluorometer
(Q32857, approx £350), which uses LED light technology, with the Qubit
Protein Quant-it kit (Q33211, £35)--life is too short to be messing
about with Bradford & Lowry assays these days. The kit each contains a
proprietary fluorescent dye, which binds specifically to proteins.
When in aqueous solution the dye emits a low fluorescence signal that
increases on binding.

All the Quant-it kits (including ones for ssDNA, dsDNA & RNA) are
compatible with reducing reagents and the presence of a wide range of
contaminants, which are listed in the back of their respective kit
manuals. The sensitivity, including the DNA/RNA kits, used with the
Qubit device are

Kit Assay range Sample
concentration
Quant-iT DNA HS Assay 0.2 - 100 ng 10 pg/mL - 100 ng/mL
Quant-iT DNA BR Assay 2 - 1000 ng 100 pg/mL - 1 mg/mL
Quant-iT RNA Assay 5 - 100 ng 250 pg/mL
- 100 ng/mL
Quant-iT Protein Assay* 0.25 - 5 mg 12.5 ug/mL - 5 mg/mL

If detergents are present, the bane of most protein assays, use the EZQ
Protein Quantitation kit R33200 which is adapted into microplate format
and involves a binding the protein to a membrane and washing to remove
contaminants, easier than it sounds.

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anurag sharma wrote:


Since we don't know what you have been up to it is difficult to crystal
ball your mistakes.

If you follow the protocoll given by Marion Bradford things should work:

@article{Bra-76,
AUTHOR= {M.M. Bradford},
TITLE= {A Rapid and Sensitive Method for the Quantitation of
Microgram Quantities of Protein Utilizing the Principles of Protein-Dye
Binding},
JOURNAL= {Anal. Biochem.},
VOLUME= {72},
YEAR= {1976},
PAGES= {248-254},
}

Important is a good quality dye (Serva (Munich) has a good reputation)
and the strict avoidance of interfering substances in the sample, most
notably detergents. Did you check the assay with samples of known
protein concentration (e.g. Immunoglobulin, do not use BSA as this gives
unusually high readings in this assay).

I just came across a nice review on the Pierce web site of these
traditional protein quantitative methods:

[Only registered users see links. ]

Dr Engelbert Buxbaum wrote:


Thanks for the info, I'll make a note of it in my protocol.

In article <[Only registered users see links. ]>, [Only registered users see links. ] wrote:

And IgG bind Comassie less than average protein. So,
it makes sense to use a 1:1 mixture of BSA and IgG
to approximate some random protein.

DK