GB/T 38477-2020.Determination of gene expression-Western blot.
1 Scope
GB/T 38477 specifies the principle, reagents or materials, instruments and equipment, determination steps and result analysis of the method for determining gene expression by protein blotting (Western blot).
GB/T 38477 is applicable to the determination of target gene expression in animal and plant tissues or in vitro cultured cells.
2 Normative references
The following documents are indispensable for the application of this document. For any dated referenced document, only the dated version applies to this document. For any undated referenced document, its latest version (including all amendments) applies to this document.
GB/T 6682 Specifications and test methods for water for analytical laboratories
3 Terms, definitions and abbreviations
3.1 Terms and definitions
The following terms and definitions apply to this document.
3.1.1
Gene expression
The process of converting the genetic information stored in DNA molecules into biologically active protein molecules through transcription and translation.
3.2 Abbreviations
The following abbreviations apply to this document.
BSA: Bovine Serum Albumin
ECL: Enhanced Chemiluminescence
SDS: Sodium Dodecyl Sulfate
SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
TBS: Tris Buffered Saline
TBST: Tween-20/ Tris Buffered Saline
4. Principle
After the proteins separated by SDS-PAGE are transferred to the transfer membrane, the primary antibody against the target protein is first reacted with the protein on the transfer membrane, and then the labeled secondary antibody is reacted with the primary antibody. Finally, the trace amount of protein is detected according to the characteristics of the secondary antibody marker.
This standard specifies the principle, reagents or materials, instruments and equipment, measurement steps and result analysis for the determination of gene expression by Western blot. This standard is applicable to the determination of target gene expression in animal and plant tissues or in vitro cultured cells. 　　

ICS07.080
National Standard of the People's Republic of China
GB/T38477—2020
Determination of gene expression
Protein blot
Determination of gene expression-Western blot2020-11-19Issued
State Administration for Market Regulation
National Administration of Standardization
Issued
Implementation on 2020-11-19
This standard was drafted in accordance with the rules given in GB/T1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. GB/T38477—2020
Drafting units of this standard: Hebei Medical University, China National Institute of Standardization, Hebei Provincial Food Inspection Institute, Agricultural Resources Research Center of Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, China Medical University, Hebei Normal University Main drafters of this standard: Lv Pin, Ma Aijin, Zhang Yan, Zhao Weidong, Cao Pengxiu, Zhao Meicheng, Wang Ning. I
1 Scope
Determination of gene expression
Protein blotting
GB/T38477—2020
This standard specifies the principle, reagents or materials, instruments and equipment, determination steps and result analysis of the method for determining gene expression by protein blotting (Western blot).
This standard is applicable to the determination of target gene expression in animal and plant tissues or in vitro cultured cells. 2
Normative references
The following documents are indispensable for the application of this document. For all dated references, only the dated version applies to this document. For any undated referenced document, the latest version (including all amendments) shall apply to this document. GB/T6682
Specifications and test methods for water for analytical laboratoriesTerms, definitions and abbreviations
3.1 Terms and definitions
The following terms and definitions shall apply to this document. 3.1.1
Gene expression
gene expression
The process of converting the genetic information stored in DNA molecules into biologically active protein molecules through transcription and translation. 2 Abbreviations
The following abbreviations shall apply to this document.
BSA: Bovine Serum Albumin ECL: Enhanced Chemiluminescence SDS: Sodium Dodecyl Sulfate SDS-PAGE: Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis
TBS: Tris Buffered Saline TBST: Tween-20/Tris Buffered Saline 4 Principle
After the proteins separated by SDS-PAGE are transferred to the transfer membrane, the primary antibody against the target protein is first reacted with the protein on the transfer membrane, and then the labeled secondary antibody is used to react with the primary antibody. Finally, the trace amount of protein is detected according to the characteristics of the secondary antibody marker 1
GB/T38477—2020
5 Reagents or materials
5.1 General
Unless otherwise specified, only analytical reagents are used. Water
GB/T6682, secondary.
31mol/L Tris-hydrochloric acid solution
Weigh 12.1g Tris and add 80mL water to dissolve it, adjust it to the required pH with hydrochloric acid, and add water to make up to 100mL. 5.42mol/L sodium chloride solution
Dissolve 117g sodium chloride in water and dilute to 1000mL. 50.5mol/L ethylenediaminetetraacetic acid solution
Weigh 18.61g disodium ethylenediaminetetraacetic acid dihydrate, add 80mL water, stir well, adjust pH to 8.0 with sodium hydroxide, and then dilute to 100mL.
6 Animal cell/tissue total protein lysate
Take 5mL 1mol/LTris-hydrochloric acid solution (pH8.0), 7.5mL 2mol/L sodium chloride solution, 1mL 0.5mol/L ethylenediaminetetraacetic acid solution, 1mL ethylphenyl polyethylene glycol, 0.1g sodium dodecyl sulfate, 1g sodium deoxybenzoate, and dilute to 100mL with water. Add protease inhibitors before use.
5.7 Plant total protein extraction kit
Contains lysate and protease inhibitors. bzxz.net
31.5mol/LTris-hydrochloric acid (pH8.8) solution5.8
Add 18.15gTris to 80mL water to dissolve, adjust pH to 8.8 with 1mol/L hydrochloric acid, and dilute to 100mL with water. 5.9
Acrylamide solution
Weigh 29g acrylamide monomer and 1g N, N'-methylenebisacrylamide, dissolve in water and dilute to 100mL, filter, place in a brown bottle, store at 4℃ away from light, and use within 2 months. 5.100.1g/mL sodium dodecyl sulfate solutionWeigh 10g sodium dodecyl sulfate, dissolve in water and dilute to 100mL, store at room temperature. 0.1g/mL ammonium persulfate solution
5.11
Weigh 1g ammonium persulfate, dissolve in water and dilute to 10mL, divide and store at -20℃. 5.12 Protein concentration determination kit
Contains protein standard BSA and dye reagent. 2
5.135×SDS loading buffer
GB/T38477—2020
Take 2.5mL1mol/LTris-hydrochloric acid (pH6.8) buffer, 2g sodium dodecyl sulfate, 1.2mLβ-mercaptoethanol, 4mL glycerol, 0.2g bromophenol blue, add water to dissolve, and dilute to 40mL. 5.145×SDS-PAGE electrophoresis buffer
Weigh 23.5g glycine and 3.775g Tris, add water to dissolve, and dilute to 250mL. When using, dilute to the use concentration according to the following ratio: take 133mL5XSDS-PAGE electrophoresis buffer, 7mL0.1g/mL sodium dodecyl sulfate solution, add water to dilute to 700mL. 5.15 Transfer buffer
Weigh 3.03g Tris, 14.4g glycine, dissolve in water, add 200mL methanol to mix, dilute to 1L, and store at 4℃. 5.16 TBS solution
Weigh 75mL 2mol/L sodium chloride solution and 10mL 1mol/LTris-hydrochloric acid (pH7.5) buffer, dilute to 1L with water. 5.17 TBST solution
Weigh 75mL 2mol/L sodium chloride solution, 10mL 1mol/LTris-hydrochloric acid (pH7.5) buffer and 1mL leaf temperature-20, dilute to 1L with water.
5.18 Blocking solution
Weigh 5g skim milk powder, dissolve in 100mL TBST solution, mix well, and prepare before use. 6
Instruments
6.1 Electrophoresis apparatus.
Transfer apparatus.
Vertical electrophoresis tank.
Analytical balance, sensitivity 0.001g.
Flat plate shaker.
Low-temperature centrifuge, maximum centrifugal force 25910g. Chemiluminescence analyzer/infrared fluorescence imaging system. Oscillator.
Vacuum dryer.
6.10
6.11
6.12
2.45mm ultra-thick filter paper.
1.5mL centrifuge tube.
Magnetic stirrer.
7 Determination steps
7.1 Extraction of total protein
Extraction of animal total protein
7.1.1
Transfer 20 mg of in vitro cultured cells or ground tissues to a 1.5 mL centrifuge tube, add 150 μL to 250 μL of pre-cooled cell lysis solution (containing protease inhibitors), shake on an oscillator for 15 seconds, ice bath for 5 minutes, repeat shaking-ice bath for 30 minutes, centrifuge at 4°C, 12000r/min for 10 minutes, and obtain the supernatant.
7.1.2 Extraction of plant total protein
Follow the instructions of the plant total protein extraction kit. 7.2
2 Determination of protein concentration
Follow the instructions of the protein concentration determination kit. 3SDS-PAGE
7.3.1 Protein denaturation
After measuring the protein concentration, calculate the volume of the solution containing 100μg of total protein as the loading volume. Take out the loading sample and put it in a centrifuge tube, add 5×SDS loading buffer to a final concentration of 1X, and boil the sample in boiling water for 3min~5min to denature the protein. 7.3.2 Gel preparation
Select the corresponding SDS-polyacrylamide gel concentration according to the relative molecular weight of the protein, see Appendix A. 7.3.3 Loading
Take the protein sample in 7.3.1 and load it. Add the standard molecular weight pre-stained protein to the adjacent teeth, and make up the volume of the remaining teeth with 1× loading buffer.
7.3.4 Electrophoresis
Select the voltage to be 8V/cm. When the dye enters the separation gel, increase the voltage to 15V/cm, and continue electrophoresis until the bromophenol blue reaches the bottom of the separation gel, and then turn off the electrophoresis source.
7.4 Transfer
7.4.1 Gel preparation
Soak the gel after SDS-PAGE in dry transfer buffer for 30 minutes. 7.4.2 Semi-dry transfer of proteins
Soak the transfer membrane and two filter papers of the same size as the transfer membrane in dry transfer buffer for 5 minutes. Install the transfer device from bottom to top: anode plate-super thick filter paper-transfer membrane-gel-super thick filter paper-cathode plate, connect the flow at 0.65mA/cm2 according to the gel area, and transfer for 1.5h~3h according to the relative molecular weight of the target protein. 7.5
Blocking
Place the transfer membrane in the blocking solution and shake at room temperature for 1h. 7.6
Immunolysis
Transfer the blocked transfer membrane to the primary antibody working solution and shake at room temperature for more than 1h or shake slowly at 4℃ overnight. Discard the primary antibody working solution, wash the transfer membrane with TBST for 3 times, 10 min each time, and finally wash the transfer membrane with TBS for 5 min. Place the transfer membrane in the secondary antibody working solution and shake it at room temperature for 1h3h. Discard the secondary antibody working solution, wash the transfer membrane with TBST for 3 times, 10 min each time, and finally wash the transfer membrane with TBS for 5 min. 4
7.7 Imaging
7.7.1
Imaging of horseradish peroxidase-labeled secondary antibody GB/T38477—2020
Take equal amounts of ECL chemiluminescent solution A and B, mix them evenly, add them to the transfer membrane, protect from light for 1min~5min, and image with a chemiluminescent instrument. 7.7.2
Imaging of fluorescently labeled secondary antibodies
Imaging using a fluorescent imaging system
Result analysis
The background of the image is clean, and a uniform and slight background outline of the transfer membrane can be observed. The target band is clear and not overexposed, which means that the target protein is expressed.
GB/T38477—2020
SDS-PAGE separation gel
Prepare according to Table A.1 according to the required concentration. Table A.1
Gel concentration
1.5 mol/L Tris-salt
acid (pH8.8))
SDS-PAGE stacking gel
(Informative Appendix)
Reagent preparation
Solutions used to prepare 10 mL of separation gels of different concentrations Solution composition
0.1 g/mL dodecyl
acrylamide solution
Prepare SDS-PAGE stacking gel according to Table A.2. Table A.2
Gel concentration
1 mol/L Tris-salt
acid (pH 6.8)
0.63
sodium sulfate solution
0.1g/mL
ammonium persulfate solution
solution for preparing 5mL5% stacking gel
solution composition
0.1g/mL dodecyl
acrylamide solution
sodium sulfate solution||tt ||0.83
0.05
0.1g/mL
Ammonium persulfate solution
0.05
Tetramethylethylenediamine
0.008
0.006
0.004
0.004
0.004
Tetramethylethylenediamine
0.005
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