The principle of Folin monophenol method to determine protein content
First, the purpose
Master the principle method of measuring protein content by Folin monophenol method, familiar with the operation of spectrophotometer
Second, the principle
The process of determining protein content by Folin monophenol method includes two steps: the first step is protein and Cu 2 + under alkaline conditions
Action to form a complex, the second step is the reduction of Folin by this complex
Reagents (phosphomolybdic acid and phosphotungstic acid reagents) produce dark blue compounds, and the color depth is directly proportional to the protein content. This method is more sensitive than the biuret method
100 times. Ammonium sulfate, glycine, reducing agents such as dithiothreitol (DTT), mercaptoethanol, etc. will interfere with the reaction.
3. Instruments and reagents
1. Instrument
(1) Spectrophotometer
(2) Water bath
(3) Test tube
(4) 8 test tubes with plugs
(5) 2 small beakers
(6) Funnel and frame
(7) Analytical balance
(8) Straw: 0.5m1 X1, 1m1X2, 5m1 X1
(9) Volumetric flask: 100m1 X 2, loml X 1
(10) Filter paper, glass rod, etc.
(11) Mortar
(12) Centrifuge, centrifuge tube
2. Reagents
(1) 0.5mol / L NaOH
(2) Reagent A
1) Weigh 1Og Na2C03, 2gNaOH and 0.25g potassium sodium tartrate, dissolve and make up to volume with distilled water
500m1.
2) After dissolving 0.5 g of copper sulfate (CuS0 4? 5H 2 O), dilute to 100m1 with distilled water.
Before each use, mix 50 parts of solution A with 1 part of solution B, which is reagent A. The expiration date of this mixture
Only 1 day, expires and expires.
(3) Reagent B: Add 100g of sodium tungstate (Na 2 WO 4 .2H 2 0
), 25g sodium molybdate (Na 2 Mo 4 .2H 2 0) and 700m1 distilled water, plus 50m1 85
% Phosphoric acid, 100m1 concentrated hydrochloric acid are fully mixed, connected to the reflux condenser, and refluxed for 10h with a small fire. After the reflux is completed, 150g of lithium sulfate is added,
50ml of distilled water and a few drops of liquid bromine, the opening continues to boil for 15min
To drive off excess bromine. After cooling, the solution is yellow (if it is still green, several more drops of liquid bromine must be added dropwise and boiling continues for 15 minutes). Then dilute to
1L, filtered, and the filtrate is placed in a brown reagent bottle and stored. When using it, add about twice the water to make the final concentration equivalent to 1 mol / L hydrochloric acid.
3. Materials
Mung bean sprouts or other plant materials.
4. Operation steps
1. Drawing of standard curve
(1) Configure standard bovine serum albumin solution: accurately weigh 0.025g on the analytical balance
Crystallized bovine serum albumin, pour into a small beaker, add a small amount of distilled water to dissolve and transfer to 100m
In the volumetric flask, the residual liquid in the beaker was rinsed several times with a small amount of distilled water. The rinse liquid was poured into the volumetric flask together, and finally made up to the mark with distilled water, and prepared into a standard egg self-quality solution, in which the concentration of bovine serum albumin was
250 ug / ml.
(2) Preparation of a series of standard bovine serum albumin solutions: take a test tube 6
Add the standard solution of bovine serum albumin and distilled water according to the table below. Then add 5ml of reagent A to each tube, place it at room temperature for 10min after mixing, then add
0.5ml reagent B, mix immediately (this step should be fast, otherwise the color will be weakened). After 30min, take the protein-free 1
Test tube No. is a control, and the solution in the other 5 test tubes is sequentially compared with a spectrophotometer at a wavelength of 5OOnm. Record the absorbance of the solution in each test tube.
Reagent 1 2 3 4 5 6
250ug / mg bovine serum protein (ml) 0 0.2 0.40.6 0.8 1.0
H 2 O (ml) 1 0.8 0.6 0.4 0.2 0
Protein content (ug) 0 50 100 150 200 250
(3) Drawing of standard curve: Draw the standard curve with absorbance as the vertical scale and bovine serum autoprotein content (ug) as the abscissa.
2. Sample determination
(1) Weigh 1g of mung bean sprout hypocotyl in a mortar, add 2m1 of distilled water, and homogenize. Transfer to a centrifuge tube and use 6m1
The mortar was washed twice with distilled water and incorporated into a centrifuge tube. Centrifuge at 4000r / min for 20min. Discard the pellet, transfer the supernatant to a volumetric flask, and bring to volume
10m1.
(2) Take 2 test tubes with stoppers, add 1ml of supernatant each, add 5m1 of reagent A respectively, mix well and place for lomin
Then, add 0.5 ml of reagent B, mix quickly, place at room temperature for 30 min, colorimetrically at a wavelength of 500 nm, and record the absorbance value.
V. Results processing
Find out the content X (ug) of the protein in the measurement solution from the standard curve, and then calculate the percentage of protein in the sample.
Six, matters needing attention
Folin reagent (reagent B) is unstable under alkaline conditions, but in this experiment the reaction occurred at pH 10, so in Folin
The reagents should be mixed immediately when reacting, otherwise the degree of color development will be weakened.
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