Thursday, January 29, 2009

Protein Assay by Bradford Method

PURPOSE
To estimate the quantity of protein present in a sample by Bradford assay.

PROCEDURE

RAW MATERIALS
1. Coomassie brilliant blue g 250
2. Orthophosphoric acid
3. Bovine serum albumin
4 96-wellplate
5. Autoclaved MilliQ water
6. Absolute alcohol (99.9% ethanol)

Preparation Of Bradford’s Reagent
Dissolve 50mg of Coomassie brilliant blue G 250 slowly in 25 ml of absolute alcohol.

Take 50 ml of Orthophosphoric acid in a burette and add it drop by drop to the dye solution kept
stirring on a magnetic stirrer.

Make up the volume to 100ml with autoclaved MilliQ water.

Store the reagent in an amber bottle at room temperature protected from light (The bulk dye can
be stored at 4 C).

Prepare fresh Bradford reagent in every 10 days.

Preparation of Protein Standard
Prepare a 1 mg/ml BSA stock in autoclaved MilliQ water. This is done by weighing out 50mg BSA into 50mL water.

Prepare a working stock from the above by diluting 1 ml to 10 ml with autoclaved MilliQ water (the final concentration should be 100ug/ml).


Protein Estimation

Pipet between 5 and 25 g of protein sample in 800 l total volume into a microfuge tube. If the approximate protein concentration is unknown, assay a range of dilutions (1,1:10, 1:100, 1:1000). Prepare duplicates of each sample.

For the standard curve, Pipet duplicate volumes of 50,100,150, 200 and 250 l of working standard solution into microfuge tubes and make each up to 800 l with autoclaved MilliQ water. Pipet 800l of water into a separate tube to make the reagent blank.

Add 200l of Bradford reagent to the blank, standard and sample tubes and make up the volume
to 1000l. Mix well by gentle vortex-mixing. Avoid foaming, which will lead to poor
reproducibility.

Pipet 200 l of each tube and put into a 96 well plate and incubate the plate at 30 C for 10
minutes and measure A 595 of the samples and standards against the reagent blank using a
spectrophotometer. The reading of the sample must lie within the standard reading. If the
reading is below the lowest standard reading, then use a lower dilution. If the reading is higher
than the highest standard reading, then use a higher dilution.

The software using the calibration curve automatically calculates the protein concentration of
unknown sample.

Calculation

The protein concentration of a sample can be calculated using the formula below:
X=Y-A
B
Where X=Concentration,Y=A 595,B=Slope and A= Intercept.

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