Instituto Fitotecnico Santa Catalina (FCAyF, UNLP)
CIGEN (UNLP, CONICET, CIC)
1. Technician of CIC 2. Technician of CONICET 3. Researcher of CONICET
A. Maize endosperms have to be milled to 100 mesh and the flours defatted with n-hexane using a ratio 1:10 (sample:solvent) at 4 C and stirring during one hour.
B. Several sample:buffer ratios were tested. The ratios examined were 40 mg flour:1000 uL buffer, 70:1000 and 100:1000.
C. Proteins were extracted from meal for 2, 6, 12 and 24 hours at room temperature using SDS-extraction buffer prepared as follows: 18.0 ml water + 7.65 ml 3x buffer + 1.35 ml 2-ME. The 3x buffer contained: 6.25 ml Tris HCl (ph: 6.8) + 12.05 ml tap water + 2.0 g SDS + 10.0 mg gamma pironine + 10.0 ml glycerol. During the extraction time, the samples were vortexed periodically.
D. Protein extracts were heated in boiling water for 10 minutes with later cooling under water flow up to room temperature.
E. The extracts were clarified by centrifugation for 10, 12 or 15 minutes at 12,500 - 13,000 and 14,000 x G.
F. 10 and 12% gels were used, so different monomer concentrations (%T) were analyzed.
G. 50 uL of each extract were loaded onto each lane of the gels. Electrophoresis was run at constant current and variable voltage (20 to 35 ampere/gel).
H. Bands were fixed using a mixture containing 440 ml tap water + 35 ml acetic acid + 25 ml methanol during 3 hours using a shaker. Later on, gel staining was done using a mixture of 500 mg Coomasie Blue R + 250 ml methanol + 200 ml tap water + 50 ml acetic acid during 3 hours using a shaker. Finally, gel decolouring was done employing a mixture of 335 ml tap water + 40 ml acetic acid +125 ml ethanol during 3 to 4 hours using a shaker and with constant changes of the mixture. Between each step, the gels were washed three times with deionized water.
After testing all the modifications listed above, it can be said that for maize endosperm proteins, the best electrophoretic results are obtained using the following procedures:
1. Better results are obtained using fine milling and defatted endosperm flours.
2. The best sample:buffer ratio was 70:1000. When 100:1000 ratio was used, the bands heaped upon themselves producing spots and changing the electrophoretic pattern.
3. The best extracts were obtained after 24 hours extraction, shaking 4 times in a vortex 40 minutes each within the period.
4. Better results are obtained using 12% gels, as they give better definition of the bands. 10% gels are not suitable as some bands do not appear and others join amongst themselves looking like spots or single bands.
5. Better results are also obtained centrifugating 15 minutes at 14,000 x G to clarify the extracts.
The maize samples used belong to different
types of endosperm: flint, opaque-2 and waxy. When SDS-PAGE was run according
to the above procedures, 19 bands could be observed. The electrophoretic
pattern varied with the maize type. Flint maizes present all the bands
except band thirteen. Bands 1,2,3,5,6 and 18 are absent in waxy and opaque-2
materials. On the other hand, band 16 is absent in opaque-2 maizes and
band 14 was not observed in waxy maizes. It can be said that polypeptide
electrophoretic pattern is suitable to differentiate amongst groups of
maize with different endosperm natures, though it does not allow distinguishing
phenotypes within the group. For this purpose, protein fractionation must
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