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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 BioRads Bradford micro-assay set up in a 96 well flat bottom plate. The standard curve requires 80g of BSA or IgG
, and you will need one standard curve for every 7 samples. Ideally, you should also assay approximately 80g 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 L 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 L in an appropriate buffer.
**Here an appropriate buffer is one that will make the final reagent concentrations (ex. SDS, Ureaect.) 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 L diluting in 60 L 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 L of standard to 40 L of 0.05% SDS and 3M Urea, so your final solution would be 80 g of BSA in 80 L 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 L 10 L 8 L 8 L 6 L 6 L 4 L 4 L 2 L 2 L 0 L 0 L
Dilution 0 L 0 L 2 L 2 L 4 L 4 L 6 L 6 L 8 L 8 L 10 L 10 L


This gives you 5 different dilutions of each sample and a standard curve from 0-10 g 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 L 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 g 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 L 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.

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Last modified on January 7, 2008 by Jonathan Meyer