Quantitation of Protein:Folin - phenol Reagent Method (Lowry Method)
【 Purpose 】 1.Study the principle and method of Lowry method to quantitate protein. 2.Master the operational method of Lowry method to quantitate protein 【 Principle 】 The principle of Lowry method is that there are tyrosine and tryptophan residues in the protein and those two amino acid residues can react with Folin-phenol reagent. The reaction process can be divided into two steps. The first step is that the peptide bonds of protein can bind with Cu 2+ to form protein- Cu 2+ compound in the basic solution. While the second step is that the Tyr or the Trp residue in the protein- Cu 2+ compound can deoxidize the phosphotungstic acid and phosphomolydic acid of the alkali phenol reagent, and then form blue product. This reaction reaches its high point after 30 minutes. In a definite range, the relationship between absorbance and protein concentration is a straight line. So we can determine protein concentration by spectroscopic method. When we use spectroscopic method, we choose different wavelength depending on different protein concentration. If the protein concentration is high (25-100ug), we need to detect under the wavelength 500nm, but if the protein concentration is low (5-25ug), we choose the wavelength 755nm.Then calculate the protein concentration of unknown sample from the calibration curve. Folin-phonel method is easy to perform and has high sensitivity when the protein in the sample weighs up more than 5ug. It is one of the most widely used methods in the quantitation of protein. 【 Apparatus 】 Test tubes and test tube shelf, 100ml flasks, Pipets, 721 type spectroscope 【 Reagents 】 1.Folin- phenol reagent A: Dissolve 1g Na 2 CO 3 into 50ml of 0.lmol/L NaOH, and then dissolve 0.5g of CuSO 4 ·5H 2 O into 100ml of 1% sodium potassium tartrate, mix 50ml of the former and 1ml of the latter. The mixer is useful in one day. 2.Folin- phenol reagent B : Add l00g of Na 2 WO 4 • 2H 2 O, 25g of Na 2 MoO 4 • 2H 2 O, 700ml of distilled water, 50ml of 85% H 3 PO 4 and 100ml of thick HC1 into a 1.5L round- bottom stoppered flask, mix well and reflux l0h. After reflux, add 150g of Li 2 SO 4 , 50ml of distilled water, several drops liquid Br 2 , boil 15min without cap to get rid of excessive Br 2 . After cooling, dilute to a constant volume of 1000ml by volumetric flask, filtrate; add several drops of bromic water to be oxidated to pale yellow while the filtrate is green. Keep the filtrate in brown flask in darkroom. Titrate with calibration NaOH before using to ascertain the acidity of the reagent, the indicator is phenolphthalein. The acidity of the reagent is about 2mol/L commonly (because the filtrate is pale yellow, dilute 100 times before titrating in order not to affect observing end - point). Dilute properly (about one time) before using to give 1 mol/L acid. 3. Standard protein solution: Accurately weigh bovine (or human) serum albumin 100mg with analytical balance, dissolved in minimum volume of distilled water, transfer to a 100 ml flask, accurately dilute to 100ml, which makes the concentration of protein 1mg/ml. 4. Sample solution: Make 0.5mg/ml casein solution as unknown solution. 【 Procedures 】 1.Draw calibration curve Number seven test tubes, add reagents as the following table, shake them up right now.
| Reagent Tube No. | 0 | 1 | 2 | 3 | 4 | 5 | 6 |
| Standard protein solution(ml) Distilled water(ml) Folin-phonel reagent A(ml) | 0 1 5 | 0.1 0.9 5 | 0.2 0.8 5 | 0.3 0.7 5 | 0.4 0.6 5 | 0.5 0.5 5 | 0.6 0.4 5 |
Mix and keep 10 minutes in room temperature
| Folin-phonel reagent B(ml) | 1 | 1 | 1 | 1 | 1 | 1 | 1 |
Add Folin-phonel reagent B, shaking immediately, keep 30 minutes the determine absorbance. Write down the absorbance at 500nm of each test tube. The test tube numbered 0 is contrast. Draw the calibration curve while the absorbance is the y-axis, the standard protein concentration is the x-axis. 2. Determine concentration of sample solution： Take two test tubes, imbibe 1ml sample solution truly, add Folin-phonel reagent A, shake , keep 10 minutes at room temperature, add 1ml Folin-phonel reagent B, shaking immediately, keep 30 minutes then determine absorbance at 500nm. 【 Results 】 According to the absorbance of unknown sample solution, check out the protein concentration in the sample solution from the calibration curve.
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