1、BRITISH STANDARD BS 4317-30: 1994 ISO 8981:1993 Methods of test for Cereals and pulses Part 30: Identification of wheat varieties by electrophoresis UDC 633.11:543.54:664.64.016.7BS4317-30:1994 This British Standard, having been prepared under the directionof the Agriculture and Food Standards Polic
2、y Committee,was published underthe authority of the Standards Board and comes into effect on 15February1994 BSI 08-1999 The following BSI references relate to the work on this standard: Committee reference AFC/4 Draft for comment 91/55374 DC ISBN 0 580 22950 5 Committees responsible for this British
3、 Standard The preparation of this British Standard was entrusted by the Agriculture and Food Standards Policy Committee (AFC/-) to Technical Committee AFC/4, upon which the following bodies were represented: Agricultural Engineers Association Association of Public Analysts British Edible Pulse Assoc
4、iation Department of Trade and Industry (National Weights and Measures Laboratory) Flour Milling and Baking Research Association Grain and Feed Trade Association Home Grown Cereals Authority Institute of Brewing Institute of Food Science and Technology Intervention Board for Agricultural Produce Min
5、istry of Agriculture, Fisheries and Food NABIM National Farmers Union Natural Resources Institute Processors and Growers Research Organization Silsoe Research Institute UnitedKingdom Agricultural Supply Trade Association Ltd. Amendments issued since publication Amd. No. Date CommentsBS4317-30:1994 B
6、SI 08-1999 i Contents Page Committees responsible Inside front cover National foreword ii Introduction 1 1 Scope 1 2 Principle 1 3 Reagents 1 4 Apparatus 2 5 Sampling 2 6 Procedure 2 7 Photography 3 8 Pattern interpretation 4 9 Expression of results 4 Annex A (normative) Confidence intervals 5 Annex
7、 B (informative) Bibliography 6 Figure 1 PAGE patterns of gliadins of Neepawa (1 and 12), Slejpner (2), Rektor (3), Prinqual (4), Mercia (5), Maris Huntsman (6), Galahad (7), Champlein (8), Camp Remy (9), Beauchamp (10) and Avalon (11) wheats 4 Table A.1 5 Table A.2 6 List of references Inside back
8、coverBS4317-30:1994 ii BSI 08-1999 National foreword This Part of BS4317 has been prepared under the direction of the Agriculture and Food Standards Policy Committee. It is identical with ISO8981:1993 Wheat Identification of varieties by electrophoresis, published by the International Organization f
9、or Standardization (ISO), and in the preparation of which the UnitedKingdom played a full part. Additional information. With reference to clause3, water conforming to grade2 of BS3978:1987 Specification for water for laboratory use is suitable. A British Standard does not purport to include all the
10、necessary provisions of a contract. Users of British Standards are responsible for their correct application. Compliance with a British Standard does not of itself confer immunity from legal obligations. Cross-references International Standard Corresponding British Standard ISO 950:1979 BS 4510:1980
11、 Methods for sampling cerals (as grain) (Identical) ISO 2170:1980 BS 5333:1981 Methods for sampling cereals and pulses (as milled products) (Identical) ISO 6644:1981 BS 6298:1982 Methods for automatic sampling of cereals and milled cereal products by mechanical means (Identical) Summary of pages Thi
12、s document comprises a front cover, an inside front cover, pagesi andii, pages1 to6, an inside back cover and a back cover. This standard has been updated (see copyright date) and may have had amendments incorporated. This will be indicated in the amendment table on the inside front cover.BS4317-30:
13、1994 BSI 08-1999 1 Introduction The protein composition of wheat results from direct genetic control and in general is not affected by environmental conditions (e.g. location or year of growth). In addition, because wheat is essentially a self-pollinating plant, the protein composition of the differ
14、ent varieties of wheat remains stable for several plant generations. Therefore, the protein composition of a wheat can be used to characterize and thus to identify its variety. Protein profiles can be obtained by carrying out polyacrylamide gel electrophoresis (PAGE) separations of the wheat gliadin
15、s. The polyacrylamide gels are stained to make the separated protein components visible. If such protein profiles are established for all wheat varieties which can be expected to occur in a particular region (i.e. a variety catalogue is prepared), the identification of an unknown variety of wheat ca
16、n be established by reference to such a catalogue. Such a practice has been thoroughly characterized and is in common use in numerous countries. 1 Scope This International Standard specifies a method for the identification of the variety of a given lot of soft or hard wheat, in the form of individua
17、l ground kernels, flour farina or semolina, by the separation of gliadin proteins. 2 Principle The separation of gliadin wheat proteins by polyacrylamide gel electrophoresis (PAGE) in slab gels containing aluminium lactate buffer, pH3,1. 3 Reagents Use only reagents of recognized analytical grade un
18、less otherwise specified. The water used shall be deionized water having a resistance greater than10M7. 3.1 Extraction solution, ethanol, 70% (V/V). Dilute 700 ml of ethanol (absolute) with 300 ml of water. 3.2 Buffer solution, aluminium lactate,2,5g/l solution. Dissolve15,0g of aluminium lactate in
19、5,5litre of water. Adjust the pH to3,1 with lactic acid. Make up to6litre with water. Vacuum filter through a filter of pore size0,454m. Store at 4 C. NOTE 1The conductivity of the solution should be approximately 9504S/m. 3.3 Sample dilution buffer Dissolve60,0g of sucrose in50ml of buffer solution
20、(3.2). Store at 4 C. 3.4 Acrylamide, 60,0 g/l gel solution. WARNING Acrylamide monomer is a neurotoxic substance which can be absorbed through the skin. Caution should be taken when handling either the crystalline powder or the gel solution. Dissolve, in a100ml one-mark volumetric flask,6,0g of acry
21、lamide (electrophoresis grade),0,300g of N,N-methylene bisacrylamide (electrophoresis grade) and0,020g of ascorbic acid in the buffer solution(3.2). Make up to the mark with the buffer solution. Store at 4 C. 3.5 Initiator solution, iron(II) sulfate heptahydrate,10g/l solution. Dissolve, in a 10 ml
22、one-mark volumetric flask,0,1g of iron(II) sulfate heptahydrate in water and make up to the mark with water. Prepare this solution shortly before use. 3.6 Catalyst solution, hydrogen peroxide, 0,99 % (m/m) solution. Dissolve, in a10ml one-mark volumetric flask,0,33ml of hydrogen peroxide 30%(m/m) in
23、 water. Make up to the mark with water. Prepare a fresh solution daily and store at 4 C. 3.7 Trichloroacetic acid, 9,6 % (m/m) solution. Dissolve, in a 1 000ml one-mark volumetric flask,96g of trichloroacetic acid in water. Make up to the mark with water. 3.8 Brilliant blue R250, 5,0 g/l stock solut
24、ion. Dissolve 5,0 g of Brilliant blue in 1 litre of ethanol, stir for 1 h, and then filter through a filter paper(4.10) to remove any inorganic salts present. 3.9 Staining solution: Brilliant blue, 0,25 g/l solution. Mix5,0 ml of stock solution(3.8) with95ml of trichloroacetic acid solution (3.7). 3
25、.10 Marker or tracking dye, e.g. Pyronin G, Methyl green.BS4317-30:1994 2 BSI 08-1999 4 Apparatus Usual laboratory apparatus and, in particular, the following. 4.1 Vertical electrophoresis unit 1) 4.2 Slot-formers, appropriate for the equipment used. 4.3 Power supply A d.c. power supply which provid
26、es stable and constant voltages up to 1000V and constant current up to 300mA (300Wmin.). 4.4 Cooling bath, thermoregulated. A circulating water bath to maintain the electrophoresis unit at20 C 1 C. 4.5 Water purification system A deionization system to provide high quality water having a resistance
27、greater than 10M7. 4.6 Analytical balance 4.7 pH-meter, with an accuracy of 0,05 pH units. 4.8 Conductivity meter (optional). 4.9 Vacuum filter unit, with cellulose nitrate filters, pore size0,45 4m. 4.10 Filter paper, Whatman No. 1, 15,0 cm. 4.11 Magnetic stirrer, with PTFE-coated 2)stirring bars,
28、2,5 cm and 5 cm long. 4.12 Hammer and steel plate, for crushing individual kernels. 4.13 Sample grinder, capable of yielding a particle size of5004m. 4.14 Sample tubes, with caps. 4.15 Micropipettes Hand-held repeater pipettes with2,5ml disposable tips to deliver volumes of504lto2504l. Adjustable mi
29、cropipettes to deliver104l to1004l and1004l to1000 4l. 4.16 Microcentrifuge, with minimum centrifugal force of10000g. 4.17 Centrifuge tubes, of capacity1,5ml2,0ml. 4.18 Vortex stirrer 4.19 Microsyringes, of capacity254l. 4.20 Rigid polyethylene containers, approximately6cm 17cm 17cm. 4.21 Pyrex glas
30、s trays, 33cm 23cm 5cm, for gel washing, viewing and photography. 4.22 Variable-speed shaker, capable of a shaking speed of approx. 50 rpm. 4.23 Light box, equipped with a fluorescent light source and a minimum viewing surface area of30cm 60cm for gel examination and photography. 4.24 Camera Single-
31、lens reflex35mm camera, loaded with fine-grain black and white or colour film. 4.25 Camera stand Any stable stand for photography with a camera using slow shutter speeds. 5 Sampling It is important that the laboratory receive a sample which is truly representative and has not been damaged or changed
32、 during transport or storage. Sampling is not part of the method specified in this International Standard. Recommended sampling methods are given in ISO950, ISO2170 and ISO6644. 6 Procedure 6.1 Preparation of test samples and extraction 6.1.1 Single kernel samples NOTE 2The number of individual seed
33、s which need to be analysed depends on the accuracy of determination required. Confidence limits based on the examination of various numbers of kernels are listed in Annex A. Analysis of larger sub-samples yields proportion estimates of greater accuracy. Place the kernel between folded paper (5cm 5c
34、m) on a metal plate and pulverize with the hammer(4.12). Transfer the crushed material to the sample tube (4.14). Add2004l of extraction solution (3.1) using a repeater pipette (4.15). Mix the contents with the vortex mixer (4.18) for 10s and allow to stand at room temperature (20 C) for at least1h.
35、 Add1004l of sample dilution buffer(3.3) and remix. Allow mixture to settle for10min before sampling for analysis. If the kernel extract is to be kept for more than24h, transfer extract into a centrifuge tube (4.17) and centrifuge for3min at 10000g in the micro-centrifuge (4.16). Decant the supernat
36、ant into a sample tube and replace the cap. Store at4 C for up to10days. NOTE 3The volumes used are for an average weight of30mg. Generally, most laboratories do not weigh the individual kernels, but, if desired, one could weigh the kernel and then add volumes equivalent to a total of 104l per milli
37、gram of wheat. Using kernel weights results in better electrophoregrams but it increases sample preparation times. 1) Numerous commercial vertical electrophoresis units are available. 2) PTFE = polytetrafluoroethyleneBS4317-30:1994 BSI 08-1999 3 6.1.2 Bulk samples Grind the sample in the electric gr
38、inder (4.13). Weigh0,2g of ground meal into a centrifuge tube(4.17) and add6004l of extraction solution. Mix the contents with the vortex mixer (4.18) for10s and allow to stand at room temperature(20 C) for at least1h. Centrifuge at10000g for5min. Transfer2004l of the supernatant into a sample tube
39、containing4004l of sample dilution buffer (3.3) and replace the cap. Mix for10s. Store at 4 C for up to 10days. 6.2 Preparation of standard extract Proceed as described in6.1 but use kernels of the pure reference variety (Neepawa) 3) . 6.3 Preparation of gels Assemble gel cassettes with1,5mm-thick s
40、pacers, as described in the manufacturers instructions. NOTE 4To aid in pouring the gel solution and inserting the slot-former into the cassette, tape a piece of plastic (e.g.Plexiglas 4) , 140mm 25mm 3mm) to the top edge of one of the cassette glass plates. To prepare each gel, combine30g of gel so
41、lution(3.4) and754l of initiator solution (3.5) in a125ml filtering flask with a side arm. Degas the solution under a vacuum of at least50mmHg for2min with constant mixing. Add1204l catalyst solution (3.6). Swirl the solution gently by hand for10s, taking care not to incorporate air bubbles. Quickly
42、 pour the solution into the cassette in a continuous stream to minimize the introduction of air. Immediately after the cassette is filled, insert the slot-former (4.2). Polymerization is complete in5min to10min. NOTE 5A casting temperature of20 C to22 C has been found to give satisfactory results. 6
43、.4 Sample loading Carefully remove the slot-former and fill the sample wells with buffer solution (3.2) to prevent dehydration of the gel. Using a microsyringe (4.19), deposit the sample extract (6.1.1 or 6.1.2) in the bottom of the wells. The amount of sample extract depends on the equipment used.
44、(The dense sample extract forms a layer under the buffer solution in the wells). Place standard extract (6.2) in any well except the two outer wells. NOTE 6As a guide, deposit a sample of24l in each well of a30-well, 1,5mm-thick gel. 6.5 Assembly of electrophoresis unit Attach the upper buffer chamb
45、er to the tops of the gel cassettes. Assemble the components of the lower chamber according to the manufacturers instructions. Place the upper chamber/gel cassette assembly into the lower chamber which has been filled with buffer solution (3.2) (or enough buffer to cover the gel cassettes). Fill the
46、 upper chamber with buffer solution, place the safety lid onto the unit and connect the electrodes to the power supply (4.3). Ensure that the upper chamber electrode is the anode and that it is connected to the positive (+) pole of the power supply, while the lower chamber electrode is the cathode a
47、nd is connected to the negative () pole of the power supply. 6.6 Electrophoresis Set the water bath (4.4) to maintain the electrophoresis unit at20 C during the run. Use a constant current of90 mA per gel for electrophoresis. Use a marker or tracking dye(3.10) to determine the optimum run-time for d
48、ifferent types of equipment and sizes of gels. When the run is finished, turn off the power supply, disconnect the safety lid, remove the upper chamber assembly and pour off the upper layer of buffer. WARNING The high voltages are potentially lethal. Ensure that the electrophoresis apparatus is used
49、 safely according to the manufacturers instructions and that no electrical leaks are present. 6.7 Staining Detach the gel cassettes from the electrophoresis unit. Open the cassettes, remove the gels and place each in a plastic container (4.20) containing100ml of staining solution (3.9). Place the covered containers on the shaker (4.22) and agitate the contents gently at approximately 50rpm for4h to18h. Destaining is not required, but place the gels in glass trays containing water and wipe them carefully with a cotton swab to remove p
