Folin- phenol method
Determination of the protein is one of the most sensitive methods. The last method is a kind of the most widely used method, because it is difficult to prepare reagent B (now can order), it has been gradually Coomassie brilliant blue method Replaced.
The color and the principle of this method. Biuret The methods are the same, just joined the second reagents, namely Folin - phenol reagent, in order to increase the amount of color, so as to improve the sensitivity of the detection of protein. These two kinds of The chromogenic reaction The reasons are: deep blue in alkaline conditions, the protein in the The peptide bond And the copper complexation. Folin - phenol reagent in phosphorus molybdate - phosphotungstic acid salt is tyrosine and tryptophan residues in the protein reduction, produces a deep blue (mixture of tungsten molybdenum blue and blue). Under certain conditions, the blue depth and proportional to the amount of protein.
Folin - phenol reagent method was first introduced by Lowry to determine the basic steps of protein concentration determination. After obtained the widespread application in the field of biochemistry. This method has the advantages of Sensitivity High, than Biuret method More sensitive to the. The disadvantage is that costs a long time, to accurately control the operation time, standard Curve It is not strictly linear form, and poor specificity, Interfering substances More. Yes Biuret reaction Interference occurs. Ion , also easy to interfere with folin- phenol reaction (Lowry reaction). But the latter's much bigger effect. Phenol, citric acid, Ammonium sulphate , Tris Buffer , Glycine , Sugar , glycerol has interference effect. Low concentration Urea 0.5-. Sulfuric acid Na (1-), sodium nitrate (1-), Three chloroacetic acid (0.5-), ethanol (5-), Ethyl ether 5-. Acetone (0.5-) solution to Color No effect, but these substances concentration is high, must be corrected Curve . Containing Ammonium sulphate The only solution, add thick Sodium carbonate - Sodium hydroxide The solution, you can color determination. If the acidity of sample is high, the color will be shallow color, you must improve Sodium carbonate - Sodium hydroxide The concentration of the solution 1~2 times.
Was measured, and F Olin phenol reagent to be especially careful, because the agent is stable only at acidic pH conditions, but the The reduction reaction Only in the case of pH=10, when the Folin is added to the phenol reagent Alkaline Copper protein solution, must mix immediately, in order to Phosphomolybdic acid - Phosphotungstic acid Before the reagent is destroyed, the reduction reaction can occur.
This method is also applicable to the quantitative determination of tyrosine and tryptophan.
This method can detect the lowest protein amounted to 5 μ G. Usually the determination range is 20 ～ 250 μ G.
(1) a (A, B) reagent:
(A Na2CO3) 10 grams, 2 grams and 0.25 grams of NaOH Potassium sodium tartrate (KNaC4H4O6 · 4H2O). Dissolved in 500 ml of distilled water.
(B) 0.5 grams of copper sulfate (CuSO4 · 5H2O) dissolved in 100 ml of distilled water, before each use, 50 (A) and 1 (B) is a hybrid, reagent.
(2) reagent b:
In 2 liters of ground return the bottle, add 100 grams Sodium tungstate (Na2WO4 · 2H2O), 25 grams Sodium molybdate (Na2MoO4 · 2H2O) and 700 ml Distilled water Add 50 ml, 85- phosphoric acid, 100 ml of concentrated hydrochloric acid, fully mixed, the reflux pipe, with small fire to reflux for 10 hour, return at the end, add in 150 grams of sulfur acid lithium (Li2SO4), 50 ml of distilled water and a few drops of liquid bromine, opening to continue boiling for 15 minutes, in order to get rid of excess bromide. After cooling the solution was yellow (such as are still green, to repeat the dropping liquid bromine steps). Diluted to 1 liters, filtration, the filtrate in Brown Reagent bottle To save. When used with a standard NaOH titration, phenolphthalein as indicator, then the appropriate dilution, about 1 times of water, so that the concentration of acid end is about 1N.
3. Standard protein solution :
Weigh accurately crystalline bovine serum albumin or G - globulin, soluble in Distilled water , concentration of about 250 mg/ml. BSA dissolved in Shuiruo opacity, can use 0.9- NaCl solution.
(1) visible spectrophotometer (2) vortex mixer (3) (4) in 16.
The 16 test tubes, 1 blank, 3 for unknown samples, the rest of the tube is divided into two groups, including 0, 0.1, 0.2, 0.4, 0.6, 0.8, 1 ml Standard protein solution (concentration of 250mg/ml). Up to 1 ml of water, then add 5 ml of reagent per test tubes a, rapid mixing in the vortex mixer, in The room temperature (20 ~ 25 ℃) for 10 minutes. Then add 0.5 ml of reagent tube B (Folin phenol reagent), also mix immediately. This step is mixed the speed to be quick, otherwise it will make the color degree of weakening. And then in the The room temperature Placed under 30 minutes, the first test tube without protein solution as a blank control group, in 700nm absorbance values in the solution was determined in each tube. The amount of protein as abscissa, the absorbance value for the vertical, draw the standard curve.
Note: for the Lowry reaction of chromogenic time continues to deepen, so the operation must be precisely controlled time: first test tubes, add 5 ml of reagent a post, the beginning of time, 1 minutes after the second test tubes, add 5 ml of reagent A, after 2 minutes with third test tubes, so on and so forth. All the test tube after the reagent a post if more than 10 minutes, the first test tubes can immediately add 0.5 ml of reagent B, 1 minutes after the second test tubes with 0.5 ml of reagent B, after 2 minutes with third test tubes, so on and so forth. To be the last one was filled with the reagent, then placed for 30 minutes, and then began to determine the optical absorption. Every minute a test sample.
Multi tube during operation, in order to prevent errors, each student must in laboratory notebooks drew the table below. The table is added to each test tube volume (ML), and from left to right, from top to bottom in order, by tube to join. The bottom two rows are the amount of protein in each tube calculated (g) and the measured absorbance value.
: Take 1 ml sample solution (of which about containing protein 20~250 micrograms), operate according to the above method, take 1 ml Distilled water Instead of samples as blank control. Determination of the sample can be usually with standard curves were put together, at the same time. In the The standard curve Determination of various behind the tube, add 3 tube. As indicated in the table in 8, 9, 10 in vitro.
According to the absorbance of the sample values, in The standard curve To identify the corresponding protein quality, so as to calculate the protein concentration of the sample solution.
Note that, because of various proteins containing different amount of Tyrosine And Phenylalanine Color depth, often with different protein changes. Thus the determination method is usually applied only to the determination of the relative concentrations of protein (relative to the standard protein).
Determination of protein content of principle of Folin- phenol method
Phosphomolybdic and phosphotungstic acid under alkaline conditions, susceptible to phenolic compounds reduction and blue reaction. The protein contains tyrosine phenol radical generation, hence the reaction sensitivity.Folin--- phenol method is 100 times the biuret method.
A: 1, 4- sodium carbonate; 2, 3, 1- 0.2N sodium hydroxide; copper sulfate; 4,2- potassium sodium tartrate. 1 and 2 mixed, 3 and 4 mixed, then two liquid mixed with 50:1 (a). That day. Two: 1NFolin-- phenol reagent (B).
The standard curve ; 0.2, 0.40, 0, 0.60, 1 ml of casein solution in a test tube (500 g / ml), add water to 1 ml, parallel to do two. According to the sequence for each 5 ml reagent (a), shake, room temperature for 10 minutes, followed by adding 1N (0.5 ml) Folin---- phenol reagent (b), shake, 30 degrees Celsius heat 30 minutes. And then in the spectrophotometric machine colorimetric assay (A500). The measured Sample And the determination of the above reactions, compared with standard curve, the results show that the content.
: phenol and citric acid have the interference on the determination, low concentration Urea , guanidine, sodium sulfate, sodium nitrate, trichloroethylene, ethanol, ethyl ether, Acetone Yes Color No effect.