HG/T 3645-1999 Determination of protein in natural rubber gloves by water extraction Lowry method HG/T3645-1999 Standard download decompression password: www.bzxz.net

ICS83.140.99
Registration No.: 4063—1999
Chemical Industry Standard of the People's Republic of China
HG/T 3645---1999
Method for the determination of agueous extractable protein in natural rubber gloves using the Lowry methodPublished on August 12, 1999
Implemented on October 1, 2000
Published by the State Administration of Petroleum and Chemical Industry
HG/T3645—1999
The formulation of this standard can change the current situation that there is no test method for the content of water-extractable protein in latex and dry rubber products produced by Chinese manufacturers, and lay a solid foundation for improving the measurement level of latex products of Chinese enterprises and earning foreign exchange through export of Chinese latex products. This standard was proposed by the Technical Supervision Department of the former Ministry of Chemical Industry of the People's Republic of my country. This standard is under the jurisdiction of the Rubber Latex Products Sub-Technical Committee of the National Rubber Standardization Committee. The main drafting unit of this standard is the Zhuzhou Rubber and Plastic Industry Research and Design Institute of the Ministry of Chemical Industry. The main drafters of this standard are Tang Shengxiu, Sheng Layun, and Li Meihui. Chemical Industry Standard of the People's Republic of China
Method for the deternination of aqueous extractable proteinin natural rubber gloves using the Lowry method
HG/T3645—1999
People who use this standard should be familiar with common laboratory operations. This standard does not mean to address all safety issues, but only involves safety issues related to this standard. It is the responsibility of the user to establish appropriate safety and health practices and ensure compliance with any national regulations. This standard requires that it be carried out by analysts with laboratory operation skills and a considerable level of expertise in a laboratory equipped with appropriate equipment. 1 Scope
This standard provides a method for determining the content of water-soluble protein in natural rubber gloves. This standard is also suitable for the determination of water-soluble protein content in other rubber products. 2 Principle
Water-soluble protein can be extracted from a buffer solution, then purified by precipitation to separate the protein from water-soluble substances that may interfere with precipitation; the purified protein is then dissolved and the protein content is determined by colorimetry using the Lowry method. 3 Definitions
This standard adopts the following definitions.
3.1 Concentration factor F is the volume ratio of the protein extract used for precipitation to the solution used to redissolve the protein precipitate. For example; 4 mL of protein extract is used for precipitation, and then 1 mL of solution is used to redissolve the precipitate, then the concentration factor F is equal to 4. 3.2 Lowry
For the purpose of this standard, the term "Lowry" is used to indicate any modified form of the original Lowry analysis method. 3.3 Protein
The proteins mentioned in this standard are water-soluble proteins and polypeptides from natural latex and its products. 4.1 Centrifuge: performance of at least 10000×g. Centrifuge tube: 50 ml and 10 mL, polypropylene tube with low protein adsorption. 4.2 Spectrophotometer and colorimetric blood.
4.3 Vortex mixer.
4.4 Micropipette, volumetric flask.
4.5 Polypropylene tube: 50 mL and 10 mL, polypropylene tube with low protein adsorption. Approved by the State Administration of Petroleum and Chemical Industry on August 12, 1999 and implemented on October 1, 2000
5 ReagentsbZxz.net
HG/T3645-1999
5. All water used shall be double distilled water, and all reagents used shall be analytical grade or equivalent reagents. 5.2 Extraction reagent: 0.02 mol/L, pH=7.4 phosphate buffer solution. 5.3 Modified Lowry Protein Assay Reagents. 5.3.1 Reagent A: Mix 10 parts of Reagent C and 0.2 parts of Reagent D, prepare on the day of the test. Reagent B: Diluted Folin's reagent (7.2 parts Folin's reagent + 2.8 parts water). 5.3.2
Reagent C: Dissolve 6 g of sodium carbonate in 100 mL of water. 5.3.4
Reagent D: Dissolve 1.5 g of copper sulfate and 3 g of sodium citrate in 100 mL of water. 5.3.5
Sodium hydroxide solution: c(NaOH) = 0.2.mol/L. 5.3.6
Sodium deoxycholate (DOOC), 0.15% (mass/volume), 0.15 g of sodium deoxycholate in 100 mL of water. Trichloroacetic acid (TCA). 72% (mass/volume), 72 g trinitroacetic acid is dissolved in 100 mL water. Pt (phosphotungstic acid) (PTA): 72% (mass/volume), 72 g tungstophosphoric acid is dissolved in 100 ml water. 5.3.8
5.3.9 Protein mother solution: Prepare a bovine serum albumin standard protein solution with a concentration of 1 mg/mL. The solution can be stored for 48 hours under refrigeration, and can be stored for 2 months at -18 ℃. When thawing, it is required to heat to 45 ℃ in a water bath and keep warm for 15 minutes. Note 1 The cooling time is the time between the curtains. In order to avoid repeated freezing and thawing, it is recommended to store the protein mother solution in several portions, each portion is sufficient to prepare a standard curve or to be used in the test steps. 6 Determination steps
6.1 Extraction of protein
6.1.1 During the whole extraction process, phosphate buffer solution with pH = 7.4 is used as the extraction medium, and its temperature should be maintained at (25±5)°C.
6.1.2 Accurately weigh (4±0.5)g of the sample (accurate to 0.0001g), cut it into pieces of 1 cmX4cm, and place it in a 50 ml polypropylene tube (4.5). Pipette the extraction reagent (5.2) into the polypropylene tube containing the test piece at a ratio of sample volume: extraction reagent volume less than or equal to 1:10, so that the test sample is evenly soaked in the extraction reagent and maintained at (25±5)°C. Extract for 2h, and stir the test piece once every 30min.
6.1.3 Pour the extract into a 50 mL centrifuge tube and centrifuge at 3000×g for 15 min. Carefully pour out the supernatant and proceed to the next step immediately. Otherwise, the supernatant must be stored at 2~8℃ for no more than 48 h. 6.2 Preparation of protein standard solution
Dilute the protein mother solution (5.3.9) with water to prepare the following series of protein standard solutions: 100μg/mL, 50ug/mL, 20g/ml., 10μg/mL. 5μg/mL, 2μg/mL. Note 2 If the protein mother solution is stored under cold conditions, it should be noted that the cumulative cold time should not exceed 2 days. 6.3 Protein precipitation and concentration
6.3.1 Perform the test at (25±5)℃ with three replicates. 6.3.2 Take 4 ml of water (blank), standard protein solution (six) and glove extract (three) and place them in 10 ml propylene centrifuge tubes, add 0.4 mL DOC (5.3.6) to each tube, mix with a vortex mixer, and let stand for 10 min at room temperature; then add 0.4 mL TCA (5.3.7) to each tube; mix with a vortex mixer, then add 0.4 mL PTA (5.3.8) to each tube, mix with a vortex mixer, and let stand for 30 min at room temperature.
6.3.3 Centrifuge the centrifuge tube (6.3.2) at 10,000× g for 30 min. Pour off the supernatant, invert the centrifuge tube on filter paper, and let the water drain.
6.3.4 Pipette the minimum volume (1.0~4.0 mL according to the amount of solid precipitate) of NaOH (5.3.5) into each centrifuge tube (6.3.3) (including the blank), mix with a vortex mixer, and redissolve the precipitate for about 20 minutes. The redissolved protein solution can be stored at (3±1)°C for 48 hours.
HG/T 3645-..1999
Note 3 If the centrifugation speed is too low, the protein precipitate may not be fully separated, resulting in erroneous conclusions. 6.4 Colorimetric test
6.4.1 Pipette 0.8 mL of the redissolved protein solution (including the blank) into each polyolefin tube (4.5), add 0.3 mL of reagent A (5.3.1) to each, mix; add 1.1 ml of reagent B (5.3.2), mix immediately with a vortex mixer. Let stand at room temperature for 30 min. 6.4.2 Within 1 h after adding reagent B, transfer 1 ml of solution (6.4.1) to the colorimetric blood and measure its absorbance at 750 nm against the blank. Take the average of the three replicate values. Note 4 During the determination process, the consistency of time, instrument and test length is very important for the test results. 7 Result Expression
7.1 Calculation
7.1.1 Standard Curve
Prepare a standard curve based on the corresponding relationship between the concentration of the protein standard solution and the absorbance. When the concentration of the protein standard solution is less than 100 g/ml, the standard curve is linear.
7.1.2 Concentration Determination
Determine the protein concentration of the sample directly from the linear part of the standard curve. 7.2 Results
The protein content in the sample (μg/g) is calculated according to the following formula: Extractable protein content (μ/g) =
Wherein: V…The volume of the extracting solution, mL,
——--The protein concentration measured from the standard curve, ug/mL; F---The concentration factor, F4 mL/volume of NaOH used to dissolve the protein precipitate, mL; m--The mass of the gloves extracted, g.
The range of the parallel results is not greater than 10g/g.
8 Test report
The test report shall at least include the following contents:
The standard adopted;
bThe name of the sample to be tested:
cThe test results;
dThe test date;
The tester.
(Beijing) Xindengzi No. 039
People's Republic of China
Chemical Industry Standard
Water Extraction Protein in Natural Rubber Gloves
Determination Method Lowry Method
HG/T 3645--1999
Published and distributed by Chemical Industry Press
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Format 88×1230mm 1/16 Printing sheet Number of characters 11 First edition in August 2000 First printing in Beijing in August 2000 Book number: 155025·0013
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