Bio-Rad Protein Assay - Manual (Page 6)

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Section 2
Instructions
2.1 Reconstituting the Standard
To reconstitute the lyophilized bovine gamma globulin and bovine
serum abumin standards, add 20 ml of deionized water and mix until dis-
solved. If the standard will not be used within 60 days, it should be
aliquoted and frozen at -20 °C.
Note: The standards contain buffer salts required for solubilizing the protein.
2.2 Standard Procedure
1. Prepare dye reagent by diluting 1 part Dye Reagent Concentrate with
4 parts distilled, deionized (DDI) water. Filter through Whatman #1
filter (or equivalent) to remove particulates. This diluted reagent may
be used for approximately 2 weeks when kept at room temperature.
2. Prepare three to five dilutions of a protein standard, which is repre-
sentative of the protein solution to be tested. The linear range of the
assay for BSA is 0.2 to 0.9 mg/ml, whereas with IgG the linear range
is 0.2 to 1.5 mg/ml. (See Common Questions, question 4, for more
information.)
3. Pipet 100 µl of each standard and sample solution into a clean, dry
test tube. Protein solutions are normally assayed in duplicate or tripli-
cate.
4. Add 5.0 ml of diluted dye reagent to each tube and vortex.
5. Incubate at room temperature for at least 5 minutes. Absorbance will
increase over time; samples should incubate at room temperature for
no more than 1 hour.
6. Measure absorbance at 595 nm.
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