【 Purpose 】 Master the principle and method of coomassie bright blue dye - binding method to quantitate protein. 【 Principle 】 Coomassie brilliant blue G - 250, for short CBG is a red dye in acidic solution and its absorbance wavelength is 465nm. When combined with protein it shows a shift in its absorption maximum from 465nm to 595nm. The absorption at 595nm is directly proportional to protein concentration in a definite range of 1 to 1000μg. So we can determine protein concentration by color matching method. Because protein-dye has high absorbance value, the sensitivity of protein quantitation could be highly improved to 1μg protein. The dye can bind with protein immediately, about 2 minutes, and this complex can be stable within 1 hour. So this method is easy to perform, quick, highly sensitive and stable. It is a widely used method in protein quantitation. 【 Materials 】 1. Apparatus spectrophotometer, Test tube, Pipet, Flask 2. Reagents (1) Standard protein solution: Weigh 10mg bovine serum albumin, dissolved in distilled water then dilute to 100ml to get the 100μg/ml solution. (2) Coomassie bright blue G-250 solution: Weigh 100mg coomassie bright blue G-250, dissolve in 50ml 95% ethanol, then add 85%(m/v) phosphate solution 100ml, finally dilute to 1000ml. This solution can be preserved for 1 month at room temperature. (3) Sample solution: Make 50μg/ml bovine serum albumin solution as sample solution. 【 Procedures 】 1. Draw calibration curve Number six clear test tubes, and add reagents as the following table.
| Number | 1 | 2 | 3 | 4 | 5 | 6 |
| Standard protein solution（ml） Distilled water（ml） Coomassie bright blue G-250 solution（ml） Protein concentration（μg） | 0.0 1.0 5 0 | 0.2 0.8 5 20 | 0.4 0.6 5 40 | 0.6 0.4 5 60 | 0.8 0.2 5 80 | 1.0 0.0 5 100 |
After adding those reagents, shake them up, keep standing for 2 minutes at room temperature. Determine absorbance at 595nm, the first tube is contrast. Make absorbance-protein concentration calibration curve, while the bovine serum albumin concentration is x-axis, the absorbance is y-axis. 2. Sample assay Imbibe 1.0ml of sample solution truly to a clear and dry test tube, add 5ml of coomassie bright blue G-250 reagent and shake up, keep standing for 2 minutes at room temperature. Use blank tube as zero, and then make color matching at 595nm. Record the absorbance. 【 Results 】 Look up concentration of sample solution on calibration curve. 【 Questions 】 1. Try to compare the advantages and disadvantages of Folin - phenol Reagent Method with Coomassie bright blue Dye - binding method. 2. Try to compare the advantages and disadvantages of Bicinchoninic Acid Method with Folin - phenol Reagent Method. 3. What is the function of adding sulphuric acid and K 2 SO 4 -CUSO 4 mixed powder while digesting sample in the micro-Kjeldahl method. 4. What is the principle of ultraviolet absorption method to quantitate protein.
