Double atom (Biuret method)

(i) Experimental principle
NH3CONHCONH3) is the product of two molecules that are heated by about 180 degrees Cand released a molecule of ammonia. In a strong alkaline solution, a purple couple is formed with CuSO4, called a double shrink reaction. Where there are two amide-based or two directly connected peptide bonds, or can pass a peptide bond connected by an intermediate carbon atom, these compounds have a double-shrink reaction.
the color of the purple ensex is directly related to the concentration of
protein
, and is independent of the protein molecular weight and the composition of
amino acids
, so it can be used to determine protein content. The measuring range is 1 to 10 mg protein. The main substances that interfere with this determination are ammonium sulfate, Tris buffer and certain amino acids.
advantage of this method is that it is faster, different proteins produce similar colors of shade, and less interference substances. The main disadvantage is poor sensitivity. Therefore, the double-shrink method is often used in the need for rapid, but not very accurate protein determination.
(ii)
Reagents
and Equipment
1. Reagents:
(1) Standard Protein Solution: With standard crystalline cow
serum
clear protein (BSA) or standard casein, with a standard protein solution of 10 mg/ml, A280 with BSA concentration 1mg/ml is 0.66 to correct its purity. If necessary, the standard protein can also be pre-determined by micro-Kay's nitrogen determination method protein nitrogen content, to calculate its purity, and then according to its purity, weighing into a standard protein solution. Bovine serum protein is made with H2O or 0.9% NaCl and casein with 0. 05N NaOH preparation.
(2) double-shrink reagent: said to be 1.50 grams of copper sulfate (CuSO4.5H2O) and 6.0 grams of potassium sacrosanate (KNaC4H4O6. 4H2O), dissolve with 500 ml of water, add 300 ml of 10% NaOH solution with stirring, dilute with water to 1 litre, and store in a plastic bottle (or in a bottle with paraffin on the inner wall). This reagent can be stored for a long time. If black precipitation appears in the storage bottle, it needs to be re-provisioned.
2. Equipment:
visible tra-photonometer, large
tube
15, vortex mixer, etc.
(iii) how to operate
.1. Determination of the standard curve: take 12 test tubes in two groups, add 0,0.2, 0.4, 0.6, 0.8, 1.0 ml of standard protein solution, fill with water to 1 ml, and then add 4 ml of double-shrink reagent. After full shaking, place at room temperature (20 to 25 degrees C) for 30 minutes, at 540nm for color measurement. Use the first test tube without a protein solution as a blank control fluid. Take the average of the two sets of measured, take the protein content as the cross-base, and the light absorption value draw the standard curve for the vertical base.
2, sample determination: take 2 to 3 test tubes, with the same method above, determine the protein concentration of unknown samples. Note that the sample concentration should not exceed 10 mg/ml.
, Folin-Phenol Reagent Method (Lowry Method)
(i) 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 difficult preparation of reagent B (now possible to order), in recent years gradually replaced by the Coomas Lianglan law. The color-showing principle of this method is the same as that of the bi-shrink method, but only by adding a second reagent, the Folin-phenol reagent, to increase the amount of color, thereby increasing the sensitivity of the detecting protein. The reason why these two color-showing reactions produce dark blue is that, under alkaline conditions, peptide bonds in proteins bind to copper to create complexes. The phosphate-phosphate-tungstenate in folin-phenol reagents is reduced by tyrosine and phenylalanine residues in proteins to produce dark blues (a mixture of tantalum and tungsten orchids). Under certain conditions, the blue depth is directly related to the amount of protein.
The Folin-Phenol reagent method was the first to determine the basic steps of protein concentration determination by Lowry. It has been widely
in the field
biochemistry in the future. The advantage of this assay method is high sensitivity, much more sensitive than the double-shrink method, the disadvantage is that it takes a long time, to accurately control the operating time, the standard curve is not a strict linear form, and less specific, more interference substances. Ions that interfere with the double-shrink reaction are equally susceptible to interference with lowry reactions. And the impact on the latter is much greater. Phenols, citric acid, ammonium sulfate, Tris buffer, glycine, sugar, glycerin, etc. all have interference. Solutions such as lower concentrations of urea (0.5%), sulfate (1%), nitrate (1%), troploroacetic acid (0.5%), ethanol (5%), ether (5%), acetone (0.5%) have no effect on color development, but when these substance concentrations are high, correction curves must be made. A solution containing ammonium sulfate can be color-showing only by the addition of a thick sodium carbonate-sodium hydroxide solution. If the acidity of the sample is high and the color will be light after color, the concentration of sodium carbonate-sodium hydroxide solution must be increased by 1 to 2 times.
when measuring, add F olin-phenol reagents to be particularly careful, because the reagent is only stable under acidic pH conditions, but the above reduction reaction only occurs in the case of pH-10, so when Folin phenol reagents added to alkaline copper-protein solution, must be immediately mixed, so that the reduction reaction can occur before the phosphate-tungsten acid reagent is destroyed.
this method is also applicable to the quantitative determination of tyrosine and tryptophan.
the minimum protein amount that can be detected by this method is up to 5 mg. The usually measuring range is 20 to 250 mg.
(ii) Reagents and equipment
.1. Reagent
(1) Reagent A:
(A) 10 g Na2CO3, 2 g NaOH and 0.25 g potassium sodium citrate (KNaC4H4O6.4H2O). Dissolved in 500 ml distilled water.
(B) 0.5 g copper sulfate (CuSO4.5H2O) dissolved in 100 ml distilled water and mixed 50 (A) with 1 serving (B) before each use, i.e. reagent A.
(2) Reagent B:
Add 100g sodium tungstenate (Na2WO4.2H2O), 25g sodium tantalum (Na2MoO4.2H2O) and 700ml distilled water, plus 50ml in a 2-litre reflow bottle 85% phosphoric acid, 100 ml thick hydrochloric acid, fully mixed, followed by a reflow tube, to a small fire reflow for 10 hours, at the end of the reflow, add 150 grams of lithium sulfate (Li2SO4), 50 ml distilled water and several drops of liquid bromine, the opening continues to boil for 15 minutes, in order to remove excess bromine. The solution is yellow after cooling (if it is still green, repeat the step of dripping liquid bromine). Diluted to 1 litre,
filtration
, the filter is stored in a brown reagent bottle. When using the standard NaOH titration, phenolic as an indicator, and then appropriate dilution, about 1 times the water, so that the final acid concentration of about 1N.
(3) standard protein solution:
precisely called crystalline bovine serum protein or g-globulin, dissolved in distilled water, concentration of about 250 mg/ml. Cow serum protein dissolved in water if cloudy, can be used 0.9 % NaCl solution between.
2. Equipment
(1) Visible tracheometer (2) vortex mixer (3) stopwatch (4) test tube 16
(iii) operating method
1. The determination of the standard curve: take 16 large test tubes, 1 blank, 3 left as unknown samples, the rest of the test tubes are divided into two groups, respectively, added 0,0.1, 0.2, 0.4, 0.6, 0.8, 1.0 ml standard protein solution (concentration 250mg/ml). Fill with water to 1.0 ml, then add 5 ml of reagent A to each test tube, mix quickly on the vortex mixer, and place at room temperature (20 to 25 degrees C) for 10 minutes. Add 0.5 ml reagent B (Folin-phenol reagent) tube by tube and mix immediately. This step blends faster, otherwise it will reduce the color display. Then placed at room temperature for 30 minutes, the first test tube without protein solution as a blank control, at 700nm to determine the absorbance value of the solution in each tube. With the amount of protein as the cross-seater and the absorbance value as the vertical base, the standard curve is drawn.
Note: Because the color of Lowry reaction deepens over time, the operation must accurately control the time, that is, the first test tube added 5 ml reagent A, start timing, 1 minute later, the 2nd test tube added 5 ml reagent A, 2 minutes later added a third test tube, the remaining and so on. If all test tubes are added to reagent A after more than 10 minutes, then the first test tube can immediately add 0.5 ml reagent B, 1 minute after the second test tube to add 0.5 ml reagent B, 2 minutes after adding the third test tube, the remaining and so on. After the last test tube has been added, leave for another 30 minutes, and then begin to measure light absorption. Take one sample per minute.
multiple test tube operations, in order to prevent errors, each student must draw the table below in advance on the lab record book. The table is the amount (milliliters) to be added to each test tube and is added tube by tube in order from left to right, top to bottom. The bottom two rows are calculated in the amount of protein (micrograms) and measured absorbent values in each tube.
2. Sample determination: take 1 ml sample solution (which contains about 20 to 250 micrograms of protein), operate according to the above method, take 1 ml distilled water instead of sample as a blank control. Usually the determination of the sample can also be carried out in the same time as the determination of the standard curve. That is, after each test tube as measured by the standard curve, an additional 3 test tubes are added. As shown in the table above 8, 9, 10 test tubes.
the protein concentration of the sample solution is calculated by detecting the corresponding protein amount on the standard curve according to the absorbance value of the sample being measured.
note that because various proteins contain different amounts of tyrosine and phenylalanine, the depth of color often varies with different proteins. Therefore, this method is usually only applicable to the determination of the relative concentration of proteins (as opposed to standard proteins). .