Dissolve proteins, mix with dye, and read O.D. after 10 min. Source: Animal Cell Culture: A Guide to Basic Techniques, Fifth Edition

Operation method

Scheme 21.4 Experiment for determination of protein content by Bradford method

Principle

Dissolve proteins, mix with dye and read O.D. after l0min.

Move

makings

Sodium dodecyl sulfate (SLS,SDS), water soluble in 3.5 mmol/L or 0.3 mol/L NaOH. koammas Brilliant Blue G-250, 0.12 mmol/L (0.01%) in 4.7 % ethanol and 85% (W/V ) phosphoric acid: 100 mg koammas Brilliant Blue was dissolved in 50 ml 95% ethanol with 100 ml 85% phosphoric acid; then diluted with 1L of water. Dilute with 1L of water.

Protein standard solution (e.g. BSAlOpg/ml); design the experiment and then make a standard curve with BSA or a similar standard protein (1 50ug/ml) at intervals of 1 to 2 months.

Operating Procedure

1. Dissolve egg white (1-20ug) or cells (about ^1X106 ) in 100ul of 3.5 mmol/L SLS or 0.3mol/L NaOH.

2. Add 100ul of Reagent Blank Control (SLS), 100ul of Protein Solution, and lOOul of BSA Standard to 3 tubes each.

3. Add lml of Kaomas blue. Mix the solution well and let it stand for 10 min.

4. At 595nm on the spectrophotometer, use the reagent blank as a control and read the test tube as the BSA standard figure.

The reagents for the Bradford method are available in kits from Bio-Rad. The kit Sulforhodamine B [Skehanetal, 1990] and the reagent N-bis(hydroxyethyl)glycine (BCA) [Smith et al., 1985] can be used to determine protein content and are sensitive for microtitration experiments. Micro -BCA kits are available from Piece (see Appendix ID). These reagents are sensitive and suitable for the detection of small numbers of cells ( ^~1X103 ).

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