GB/T 38477-2020 English PDFUS$189.00 · In stock
Delivery: <= 3 days. True-PDF full-copy in English will be manually translated and delivered via email. GB/T 38477-2020: Determination of gene expression - Western blot Status: Valid
Basic dataStandard ID: GB/T 38477-2020 (GB/T38477-2020)Description (Translated English): Determination of gene expression - Western blot Sector / Industry: National Standard (Recommended) Classification of Chinese Standard: A21 Classification of International Standard: 07.080 Word Count Estimation: 10,155 Date of Issue: 2020-11-19 Date of Implementation: 2020-11-19 Regulation (derived from): National Standard Announcement No. 26 of 2020 Issuing agency(ies): State Administration for Market Regulation, China National Standardization Administration GB/T 38477-2020: Determination of gene expression - Western blot---This is a DRAFT version for illustration, not a final translation. Full copy of true-PDF in English version (including equations, symbols, images, flow-chart, tables, and figures etc.) will be manually/carefully translated upon your order.Determination of gene expression-Western blot ICS 07.080 A21 National Standards of People's Republic of China Western Blotting 2020-11-19 released 2020-11-19 implementation State Administration for Market Regulation Issued by the National Standardization Management Committee ForewordThis standard was drafted in accordance with the rules given in GB/T 1.1-2009. This standard was proposed and managed by the China National Institute of Standardization. Drafting organizations of this standard. Hebei Medical University, China National Institute of Standardization, Hebei Food Inspection and Research Institute, Chinese Academy of Sciences Genetics and Development Agricultural Resources Research Center, Institute of Educational Biology, China Medical University, Hebei Normal University. The main drafters of this standard. Lu Pin, Ma Aijin, Zhang Yan, Zhao Weidong, Cao Pengxiu, Zhao Meicheng, Wang Ning. Western Blotting1 ScopeThis standard specifies the principles, reagents or materials, instruments, equipment, and measurement methods for determining gene expression by Western blotting. Determine the steps and analysis of the results. This standard applies to the determination of target gene expression in animal and plant tissues or in vitro cultured cells.2 Normative referencesThe following documents are indispensable for the application of this document. For dated reference documents, only the dated version applies to this article Pieces. For undated references, the latest version (including all amendments) applies to this document. GB/T 6682 Analytical laboratory water specifications and test methods 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 transcribing and translating the genetic information stored in DNA molecules into protein molecules with biological activity. 3.2 Abbreviations The following abbreviations apply to this document. BSA. Bovine Serum Albumin (Bovine Serum Albumin) ECL. Enhanced Chemiluminescence (Enhanced Chemiluminescence) SDS. Sodium Dodecyl Sulfate SDS-PAGE. Sodium Dodecyl Sulfate-Polyacrylamide Gel Electrophoresis Electrophoresis) TBS. Tris Buffered Saline (TrisBufferedSaline) TBST. Tween-20/T ris Buffered Saline (Tween-20/T risBufferedSaline)4 PrincipleAfter the proteins separated by SDS-PAGE are transferred to the transfer membrane, first use the primary antibody against the target protein to react with the protein on the transfer membrane. Then use the labeled secondary antibody to react with the primary antibody, and finally detect the trace protein according to the characteristics of the secondary antibody marker.5 Reagents or materials5.1 General Unless otherwise specified, only use analytical reagents. 5.2 Water GB/T 6682, level two. 5.3 1mol/LTris-hydrochloric acid solution Weigh 12.1g Tris and add 80mL water to dissolve, adjust to the required pH with hydrochloric acid, and add water to make the volume up to 100mL. 5.4 2mol/L sodium chloride solution Dissolve 117g of sodium chloride in water and dilute to 1000mL. 5.5 0.5mol/L ethylenediaminetetraacetic acid solution Weigh 18.61g disodium ethylenediaminetetraacetic acid dihydrate, add 80mL water, stir well, adjust the pH to 8.0 with sodium hydroxide, then Then dilute to 100mL. 5.6 Animal cell/tissue total protein lysate Take 5mL1mol/LTris-hydrochloric acid solution (pH8.0), 7.5mL2mol/L sodium chloride solution, 1mL0.5mol/L ethylenediamine Tetraacetic acid solution, 1 mL ethyl phenyl polyethylene glycol, 0.1 g sodium lauryl sulfate, 1 g sodium deoxycholate, and dilute to 100 mL with water. Make Add protease inhibitor before use. 5.7 Plant total protein extraction kit Contains lysis buffer and protease inhibitors. 5.8 1.5mol/LTris-hydrochloric acid (pH8.8) solution Add 18.15g Tris to 80mL water to dissolve, adjust the pH to 8.8 with 1mol/L hydrochloric acid, and add water to make the volume up to 100mL. 5.9 Acrylamide solution Weigh 29g acrylamide monomer and 1gN,N'-methylene bisacrylamide, add water to dissolve and dilute to 100mL, filter, and place in brown The bottle should be stored at 4℃ and protected from light. Use within 2 months. 5.10 0.1g/mL sodium lauryl sulfate solution Weigh 10g sodium lauryl sulfate, add water to dissolve and dilute to 100mL, store at room temperature. 5.11 0.1g/mL ammonium persulfate solution Weigh 1g of ammonium persulfate, add water to dissolve and dilute to 10mL, after aliquoting, store at -20℃. 5.12 Protein concentration determination kit Contains protein standard BSA and dye reagents. 5.13 5×SDS loading buffer Take 2.5mL 1mol/LTris-hydrochloric acid (pH6.8) buffer, 2g sodium lauryl sulfate, 1.2mL β-mercaptoethanol, 4mL glycerol Oil and 0.2g bromophenol blue are added to water to dissolve, and the volume is adjusted to 40mL. 5.14 5×SDS-PAGE running buffer Weigh 23.5g glycine and 3.775g Tris, add water to dissolve, and dilute to 250mL. Use the following proportion to dilute to use thick Degree. Take 133mL of 5×SDS-PAGE electrophoresis buffer, 7mL of 0.1g/mL sodium lauryl sulfate solution, add water to make up to 700mL. 5.15 Transfer buffer Weigh 3.03g Tris, 14.4g glycine, add water to dissolve, then add.200mL methanol to mix, dilute to 1L, store at 4℃. 5.16 TBS solution Measure 75mL of 2mol/L sodium chloride solution and 10mL of 1mol/LTris-hydrochloric acid (pH7.5) buffer, add water to make the volume to 1L. 5.17 TBST solution Measure 75mL 2mol/L sodium chloride solution, 10mL 1mol/LTris-hydrochloric acid (pH7.5) buffer and 1mL Tween-20, add Set the volume of water to 1L. 5.18 Sealing fluid Weigh 5g of skimmed milk powder, dissolve it in 100mL TBST solution, mix well, and use it now.6 Equipment6.1 Electrophoresis instrument. 6.2 Transfer membrane instrument. 6.3 Vertical electrophoresis tank. 6.4 Analytical balance, with a sense of 0.001g. 6.5 Plate shaker. 6.6 Low-temperature centrifuge, with a maximum centrifugal force of 25910g. 6.7 Chemiluminescence instrument/infrared fluorescence imaging system. 6.8 Oscillator. 6.9 Vacuum dryer. 6.10 2.45mm ultra-thick filter paper. 6.11 1.5mL centrifuge tube. 6.12 Magnetic stirrer.7 Measurement procedure7.1 Extraction of total protein 7.1.1 Extraction of total animal protein Transfer 20 mg of in vitro cultured cells or ground tissue to a 1.5 mL centrifuge tube, and add 150 μL to 250 μL of pre-cooled cell lysis Solution (containing protease inhibitor), shake 15s in a shaker, 5min in ice bath, repeat shaking-after 30min in ice bath, centrifuge at 4℃, 12000r/min 10min, get the supernatant. 7.1.2 Extraction of total plant protein Follow the instructions of the plant total protein extraction kit. 7.2 Determination of protein concentration Follow the instructions of the protein concentration determination kit. 7.3 SDS-PAGE 7.3.1 Protein Denaturation After measuring the protein concentration, calculate the volume of the solution containing 100μg of total protein as the sample amount. Take out the loaded sample in the centrifuge tube, add 5×SDS loading buffer to a final concentration of 1×, boil the sample in boiling water for 3min~5min to denature the protein. 7.3.2 Glue Select the corresponding SDS-polyacrylamide gel concentration according to the relative molecular weight of the protein, see Appendix A. 7.3.3 Sample loading Take the protein sample of 7.3.1 and load the sample, add the standard molecular weight pre-stained protein to the adjacent dentition, and make up the free dentition with 1× loading buffer volume. 7.3.4 Electrophoresis Choose a voltage of 8V/cm. When the dye enters the separation gel, increase the voltage to 15V/cm, and continue electrophoresis until the bromophenol blue reaches separation. Glue the bottom, then turn off the power. 7.4 Transfer film 7.4.1 Gel preparation Soak the gel after SDS-PAGE in the transfer buffer for 30 minutes. 7.4.2 Semi-dry transfer of protein Soak the transfer film and two filter papers of the same size as the transfer film in the transfer buffer for 5 minutes. Install the transfer device from bottom to top. Anode plate-ultra-thick filter paper-transfer film-gel-ultra-thick filter paper-cathode plate, according to the gel area, the current is turned on at 0.65mA/cm2, root According to the relative molecular weight of the target protein, transfer 1.5h to 3h. 7.5 Closed Place the transfer membrane in the blocking solution and shake at room temperature for 1h. 7.6 Immune response Transfer the sealed transfer membrane to the working solution of the primary antibody and shake at room temperature for more than 1 hour or slowly at 4°C overnight. Abandon the first antibody As a solution, the transfer membrane was washed 3 times with TBST shaking for 10 minutes, and finally the transfer membrane was washed with TBS shaking for 5 minutes. Place the transfer film in the working solution of the secondary antibody and shake at room temperature for 1h~3h. Discard the working solution of the secondary antibody, shake the transfer membrane with TBST 3 times, 10 minutes each time, and finally shake the transfer membrane with TBS for 5 minutes. 7.7 Imaging 7.7.1 Imaging of horseradish peroxidase labeled secondary antibody Take equal amounts of ECL chemiluminescent liquid A and B and mix them, add them to the transfer film, avoid light for 1min to 5min, and image with chemiluminescence instrument. 7.7.2 Imaging of fluorescently labeled secondary antibodies Use fluorescence imaging system for imaging.8 Result analysisThe imaging picture has a clean background, and the uniform slight background outline of the transfer film can be observed. The target band is clear and not exposed, which can be judged as the purpose Protein. ......Tips & Frequently Asked Questions:Question 1: How long will the true-PDF of GB/T 38477-2020_English be delivered?Answer: Upon your order, we will start to translate GB/T 38477-2020_English as soon as possible, and keep you informed of the progress. The lead time is typically 1 ~ 3 working days. The lengthier the document the longer the lead time.Question 2: Can I share the purchased PDF of GB/T 38477-2020_English with my colleagues?Answer: Yes. The purchased PDF of GB/T 38477-2020_English will be deemed to be sold to your employer/organization who actually pays for it, including your colleagues and your employer's intranet.Question 3: Does the price include tax/VAT?Answer: Yes. 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