蛋白质含量测定法.doc（蛋白质含量测定法.doc）

1、蛋白质含量测定法.doc（蛋白质含量测定法.doc）Appendix protein assayFirst Kjeldahl method1 precipitation with tungstic acidThe total protein content of the protein in the filtrate of the sample removed by the precipitation of tungstate and the content of non protein nitrogen in the filtrate were calculated.Preparation

2、of test sample solution (1) determination of total nitrogen, preparation of solution, precision sample, test sample 1ml, accurate dilution to Sodium Chloride Physiological Solution with nitrogen content of about 1mg per 1ml.(2) the preparation of non protein nitrogen determination precision, take th

3、e test product 2ml, add water 14ml, 10% sodium tungstate solution 2ml, sulfuric acid solution (1.86 to 100) 2ml, shake it well, stand aside for 30 minutes to filter, take the filtrate determination.The total nitrogen content in the tested product was determined by the precision method, the total nit

4、rogen determination solution 1ml, the Kjeldahl flask, and the nitrogen determination method (Appendix VI, A). At the same time blank control.The non protein nitrogen determination solution 5ml was used to determine the non protein nitrogen content of the tested products in the Kjeldahl flask and the

5、 nitrogen determination method (Appendix VI, A).Press down calculationCTN (mg/ml) = (VX1-V0) * c * 14.01 * n * 2_V1CNPN (mg/ml) = (VX2-V0) * c * 14.01 * n * 2_V2CPN (mg/ml) =CTN-CNPNProtein content of the test sample (%, g/ml) =CPN * 6.25 * 100_One thousandCTN was the test sample in the formula, and

6、 the total nitrogen content of solution was mg/ml;CNPN was the test solution, non protein nitrogen content, mg/ml;VX1 as a test product, total nitrogen was measured, the solution consumed, and the volume of acid titration liquid, ml;VX2 as a test sample, non protein nitrogen determination, solution

7、consumption, acid titration volume, ml;V0 is the blank test consuming the volume of acid titration liquid, ml;C is the concentration of sulfuric acid titration solution, mol/L;CPN as the test sample, protein nitrogen content, mg/ml;N dilution times for the test sample;V1 as a test product, total nit

8、rogen was measured by the volume of the solution, ml;V2 as a test sample, the volume of non nitrogen determination solution, ml;14.01 is the relative atomic mass of nitrogen;6.25 is constant (1g nitrogen is equivalent to 6.25g protein).note (1) the sample protein content of more than 10% (g/ml), in

9、addition to protein should be appropriate to increase the sample dilution, 10% sodium tungstate solution and sulfuric acid solution was correspondingly increased in proportion, the acid concentration in the solution remained 1%.(2) the determination of total nitrogen and non protein nitrogen can be

10、made by the same blank control.2, three chloroacetic acid precipitation (Xin Zeng)In this law, the content of the protein in the precipitate was determined by precipitation of three chloroacetic acid. The content of protein was calculated.Take the appropriate sample volume for the determination of t

11、he exact amount (each containing 1ml protein 4-10mg) in the appropriate bottom tip of the centrifugal tube, with an equal volume of 12% three chloroacetic acid (12 - 100) mixing, after standing for 30 minutes, centrifugal (4000 rpm), Kami Kiyo, with about 3ml water the fractional precipitation will

12、be washed into the Kjeldahl flask, as nitrogen determination method (Appendix VI A) test for the determination of total nitrogen content in the product, at the same time as blank control.Press down calculation:(VX-V0) * C * 14.01 * 2Test sample protein content (mg/ml) = - - - - - * 6.25VVX is the te

13、st sample in the formula, and the volume of acid titration liquid is consumed, ml;V0 is the blank test consuming the volume of acid titration liquid, ml;C is the concentration of sulfuric acid titration solution, mol/L;V is the volume of the sample to be tested, ml.Second law Lowry methodThe method

14、is applied to the determination of trace proteins. The protein can form copper protein complexes in alkaline solution, the compound with phenol reagent, produce blue compound, the compound blue absorbance at 650nm and protein content is proportional to the absorbance of the sample according to the c

15、alculation, the protein content of the sample.Reagents (1) phenol reagent from sodium tungstate (Na2WO4 - 2H2O) 100g, sodium molybdate (Na2MoO4 - 2H2O) 25g, the 1500ml of distilled bottle, add 700ml water, 85% 50ml phosphoric acid, hydrochloric acid 100ml, even on the return pipe (rubber plug using

16、a cork or foil package) and boiling reflux for 10 hours. Remove the reflux tube and add a few drops of lithium sulfate, 150g, water, 50ml, and bromine. Boil for about 15 minutes, remove excess bromine. Cool, add water to 1000ml, filter, as a phenolic reagent stock.The phenol reagent stock solution w

17、as titrated with sodium hydroxide solution (0.5mol/L), and then diluted with water to the concentration of 1mol/L hydrochloric acid.(2) alkaline copper solution was obtained by 0.1mol/L potassium tartrate solution and 0.04mol/L copper sulfate solution, each 0.5 ml, 4% sodium carbonate solution and 0

18、.8% sodium hydroxide solution, each 25 ml, shake, that is. This fluid should be prepared for use.(3) sodium hydroxide titration solution (0.5mol/L) of the preparation and calibration of sodium hydroxide amount, add water and stir to dissolve into a saturated solution, after cooling, polyethylene pla

19、stic bottle, a few days after clarification, clarification, the saturated solution of sodium hydroxide 28ml, add new boiled water after cooling, the 1000ml strain.In the benchmark phthalate 105 DEG C dried to constant weight of two potassium hydrogen phthalate is about 3G, precision said, cold water

20、 50ml, add new boiling shake, to minimize dissolution; plus 2 drops of phenolphthalein indicator, with the droplets near the end point; when the two phthalic acid potassium hydrogen completely dissolved, titration solution to pink. Each 1ml of sodium hydroxide solution is equivalent to 102.1mg of po

21、tassium hydrogen phthalate two. According to the consumption of this liquid and the dosage of potassium hydrogen phthalate two, the concentration of this liquid is calculated.Standard protein solution for providing a standard Human Albumin water, diluted to quantitative containing 1ml per 1mg, as a

22、stock solution. Precise amount of stock solution of 2.5ml into 25ml volumetric flask, dilute with water to the scale, shake, is the standard protein solution containing 1ml per 100 g.A certain volume of sample determination precision (protein containing about 50 g) in a test tube, add water to 1ml,

23、adding alkaline solution of copper 5ml, shake, at room temperature for 10 minutes, quickly add 0.5 ml phenol reagent, shaken at room temperature for 30 minutes, color, light UV visible spectrophotometry pectrophotometry (Appendix II A),The absorbance was measured at the wavelength of 650nm (after co

24、lor, if turbidity was found, 15 minutes after 3000 rpm, then supernatant was determined). The precise amount of standard protein solution 0.2ml, 0.4ml, 0.6ml, 0.8ml and 1.0ml were placed in the test tube, and the same method was used when the water was added to 1ml. Accurate water intake 1ml, since

25、the addition of alkaline copper solution 5ml, the same method of operation, as blank control. The linear regression equation of the protein concentration corresponding to the absorbance of the standard curve was obtained. The absorbance of the tested product was substituted into the linear regressio

26、n equation, and the protein content of the tested product was obtained.The third method of biuret methodThe law on the basis of protein peptide bond formation red complex with Cu2+ in alkaline solution, the protein content is proportional to the depth and color, using standard protein solution as co

27、ntrol, was determined by UV Vis and sample protein content by spectrophotometry.Biuret reagent kit. Take copper sulfate (CuSO4. 5H2O) 3.0g, sodium potassium tartrate (KNaC4H4O6 ? 4H2O) 9.0g, potassium iodide 5.0g, sodium hydroxide 24g, water dissolved and diluted to 1000ml, shake, that is.Standard protein solution preparation, precision, take the amount of Human Albumin standard amount, and water is diluted into 1ml 50mg solution.The preparation of