Determining Protein Concentration
by Modified Micro-Bradford Assay
The Modified Micro-Bradford Assay
Note: This assay protocol is intended
to quantify protein samples containing any reagents. It has been adapted
from the Bio-Rad Bradford protein assay kit (#500-0006). For a list
of directly compatible reagents see the BioRad Bradford assay technical
manual (technical note 1069). This protocol only addresses issues
of protein quantification.
Based on the composition of your sample buffer (against the compatibility
chart: Appendix A) chose whether to perform the standard or modified
Bradford assay as outlined below.
The Standard Bradford:
This procedure is an adaptation of BioRad’s Bradford micro-assay –
set up in a 96 well flat bottom plate. The standard curve requires
80µg of BSA or IgG, and you will need one standard
curve for every 7 samples. Ideally, you should also assay approximately
80µg of each unknown sample in order to obtain the
most data points, but this assay will be able to determine final protein
concentration as long as 0.2-5 mg/mL.
| 1 |
Perform
the protein assay in according to the standard Bradford methodologies
above. |
| 2 |
Select a volume
of your unknown protein samples to assay. Make up to 80
µL
with dH2O, or appropriate compatible buffer.
**Here you have to ensure that the final concentration of SDS
is <0.05% and Urea is <6M** |
| 3 |
Take 80 ug
of standard and also make up to 80 µL in an
appropriate buffer.
**Here an appropriate buffer is one that will make the final
reagent concentrations (ex. SDS, Urea…ect.) the same as the
unknown protein samples.
For example, if you
had a sample in 0.1% SDS, 6M urea, and you chose to assay 20
µL
– diluting in 60 µL of dH2O – your final concentrations are 0.025% SDS and 1.5M urea.If your standard was BSA at 2 mg/mL in dH2O
or weak buffer, you would need to add 40
µL of standard to 40
µL
of 0.05% SDS and 3M Urea, so your final solution would be 80
µg
of BSA in 80
µL
of 0.025% and 1.5M urea. |
| 4 |
You also need
to make a dilution buffer with components equivalent to your
unknown protein samples and standard ~ 1 mL per 96 well plate.
If your samples were prepared according to the above example,
your dilution buffer would also be 0.025% SDS and 1.5M Urea.
|
Column:
| |
1 |
2 |
3 |
4 |
5 |
6 |
7 |
8 |
9 |
10 |
11 |
12 |
|
Sample |
10 µL |
10 µL |
8 µL |
8 µL |
6 µL |
6 µL |
4 µL |
4 µL |
2 µL |
2 µL |
0 µL |
0 µL |
|
Dilution |
0 µL |
0 µL |
2 µL |
2 µL |
4 µL |
4 µL |
6 µL |
6 µL |
8 µL |
8 µL |
10 µL |
10 µL |
This gives you 5 different
dilutions of each sample and a standard curve
from 0-10 µg per well, all in duplicate.
| 5 |
Set-up each row of the 96-well plate as follows, using
one row per standard/sample: |
| 6 |
To develop assay add 200 µL of 1:5 diluted
Bradford reagent (BioRad 500-0006) to each well, mix, and
allow 5-10 min at room temperature for development. |
| 7 |
Within 60 min
of development read the plate at 595nm.
|
The Modified Bradford:
This procedure is intended for protein preparations containing reagents
at concentrations that are incompatible with the Bradford reagent.
However, all samples – including the 80 µg of BSA or IgG used
for the standard curve - need to be precipitated and resolubilized
in a compatible buffer before the assay.
| a |
Protein precipitation
(See the protocol
on protein precipitation). |
| b |
Protein Resolubilization:
- Add 80 µL of resolubilization buffer
to each protein precipitate.
- Resolubilization buffer – 0.05% SDS
and 6M Urea.
- The sample can be quickly heated to help solvation.
- Centrifuge 15000g X 5min and check for a pellet to ensure
full protein resuspension.
Perform the protein assay in according to the standard Bradford
methodologies above.
TOP |
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