First, the experimental principle
This protein assay is one of the most sensitive methods. In the past, this method was the most widely used method. Due to the difficulty in preparing the reagent B (which is now available for order), it has gradually been replaced by the Coomassie brilliant method in recent years. The color development principle of this method is the same as that of the biuret method, except that the second reagent, the folin-phenol reagent, is added to increase the coloration amount, thereby improving the sensitivity of detecting the protein. The reason why these two color reaction reactions produce dark blue color is that under alkaline conditions, peptide bonds in proteins combine with copper to form a complex. The phospholipidate-phosphonium tungstate in folin-phenol reagent is reduced by tyrosine and phenylalanine residues in the protein to produce a dark blue color (a mixture of molybdenum blue and tungsten blue). Under certain conditions, the blue depth is proportional to the amount of protein.
The folin-phenol reagent method was first determined by lowry to determine the basic steps of protein concentration determination. It has been widely used in the field of biochemistry. The advantage of this assay is that it has high sensitivity and is much more sensitive than the biuret method. The disadvantage is that it takes a long time to accurately control the operation time. The standard curve is not strictly linear, and the specificity is poor. many. Ions that interfere with the biuret reaction also easily interfere with the lowry reaction. And the impact on the latter is much greater. Phenols, citric acid, ammonium sulfate, tris buffer, glycine, sugars, glycerol, etc. all have interference effects. Lower concentration urea (0.5%), sodium sulphate (1%), sodium nitrate (1%), trichloroacetic acid (0.5%), ethanol (5%), ether (5%), acetone (0.5%), etc. The solution has no effect on color development, but when the concentration of these substances is high, a calibration curve must be made. The solution containing ammonium sulfate can be determined by coloring only by adding concentrated sodium carbonate-sodium hydroxide solution. If the acidity of the sample is high and the color will be light after color development, the concentration of sodium carbonate-sodium hydroxide solution must be increased by 1 to 2 times.
When performing the measurement, special care should be taken when adding the folin-phenol reagent because the reagent is stable only under acidic pH conditions, but the above reduction reaction occurs only at ph=10, so when the folin-phenol reagent is added to the alkaline In the copper-protein solution, it must be mixed immediately so that the reduction reaction can occur before the phosphomolybdate-phosphoric acid reagent is destroyed.
This method is also applicable to the quantitative determination of tyrosine and tryptophan.
The minimum amount of protein detectable by this method is 5 mg. Usually the measurement range is 20~250mg.
Second, reagents and equipment
Reagent
1 Reagent A:
(a) 10 g of Na 2 CO 3 , 2 g of NaOH and 0.25 g of sodium potassium tartrate (KNaC 4 H 4 O 6 -4H 2 O). Dissolved in 500 ml of distilled water.
(b) 0.5 g of copper sulfate (CuSO 4 -5H 2 O) was dissolved in 100 ml of distilled water, and 50 parts (a) and 1 part (b) were mixed before each use to obtain a reagent A.
2 Reagent B: In a 2 liter grinding reflow bottle, add 100 g of sodium tungstate (Na 2 WO 4 -2H 2 O), 25 g of sodium molybdate (Na 2 MOO 4 -2H 2 O) and 700 ml of distilled water. Add 50 ml of 85% phosphoric acid, 100 ml of concentrated hydrochloric acid, mix well, connect to the reflux tube, and reflux for 10 hours on low heat. At the end of the reflux, add 150 g of lithium sulfate (Li 2 SO 4 ), 50 ml of distilled water and a few drops. Liquid bromine, the opening continues to boil for 15 minutes to drive off excess bromine. After cooling, the solution is yellow (if it is still green, the step of adding liquid bromine is repeated). Dilute to 1 liter, filter, and store the filtrate in a brown reagent bottle. When using, it is titrated with standard NaOH, phenolphthalein is used as an indicator, and then diluted appropriately, about 1 time of water is added, so that the final acid concentration is about 1n.
3 standard protein solution: accurately weigh crystal bovine serum albumin or g-globulin, dissolved in distilled water, the concentration is about 250mg/ml. If bovine serum albumin is dissolved in water, it can be changed to 0.9% nacl solution.
2. Equipment
1 visible light spectrophotometer
2 vortex mixer
3 stopwatch
4 test tubes 16
Third, the operation method
1. Determination of standard curve: Take 16 large test tubes, 1 for blank, 3 for unknown samples, and the other tubes into two groups, adding 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ml standard protein solution respectively. (Concentration 250 mg/ml). Make up to 1.0 ml with water, then add 5 ml of reagent A to each tube, mix rapidly on a vortex mixer, and let stand at room temperature (20-25 ° C) for 10 minutes. Then add 0.5 ml of reagent B (folin-phenol reagent) one by one, and mix immediately. This step is faster to mix, otherwise the degree of color development will be weakened. Then, it was allowed to stand at room temperature for 30 minutes, and the first tube without the protein solution was used as a blank control, and the absorbance value of the solution in each tube was measured at 700 nm. The standard curve is drawn by taking the amount of protein as the abscissa and the absorbance value as the ordinate.
Note: Because the color of the lowry reaction is deepening with time, the operation must be precisely controlled. The first tube is added with 5 ml of reagent A, and the time is started. After 1 minute, the second tube is added with 5 ml of reagent A. After 2 minutes, add the third test tube, and so on. If all the tubes have been added for more than 10 minutes after the reagent A is added, the first tube can be immediately added with 0.5 ml of reagent B. After 1 minute, the second tube is added with 0.5 ml of reagent B. After 2 minutes, the third tube is added. analogy. After the last tube was added to the reagent, it was left for another 30 minutes, and then the light absorption was measured. One sample is taken every minute.
In order to prevent errors during the multi-tube operation, each student must pre-draw the following form on the experimental notebook. The table is the amount (ml) to be added to each tube, and is added tube by tube from left to right, top to bottom. The bottom two rows are the calculated amount of protein (micrograms) per tube and the measured absorbance value.
| Pipe number | 1 | 2 | 3 | 4 | 5 | 6 | 7 | 8 | 9 | 10 |
|---|---|---|---|---|---|---|---|---|---|---|
| Standard protein (250mg/ml) | 0 | 0.1 | 0.2 | 0.4 | 0.6 | 0.8 | 1.0 | |||
| Unknown protein (about 250mg/ml) | 0.2 | 0.4 | 0.6 | |||||||
| Distilled water | 1.0 | 0.9 | 0.8 | 0.6 | 0.4 | 0.2 | 0 | 0.8 | 0.6 | 0.4 |
| Reagent A | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 | 5.0 |
| Reagent B | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 | 0.5 |
| The amount of protein in each tube (mg) | ||||||||||
| Absorbance value (a700) |
2. Determination of the sample: Take 1 ml of the sample solution (containing about 20 to 250 μg of protein), and operate according to the above method, and take 1 ml of distilled water instead of the sample as a blank control. Usually the determination of the sample can also be carried out simultaneously with the determination of the standard curve. That is, after the tubes of the standard curve were measured, three more tubes were added. 8, 9, 10 tubes in the above table.
Based on the absorbance value of the sample to be measured, the corresponding amount of protein is detected on the standard curve to calculate the protein concentration of the sample solution.
Note: Since various proteins contain different amounts of tyrosine and phenylalanine, the depth of coloration often varies with different proteins. Thus the assay is generally only suitable for determining the relative concentration of a protein (relative to a standard protein).
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