In vivo pulse labeling of proteins in attached leaves with radioactive amino acids.
The pulse labeling of proteins in attached leaves by the incorporation of radioactive amino acids can be a valuable approach to study a wide range of topics pertaining to plant gene expression. Transient changes in the rate of net protein synthesis can be measured since pulse labeling permits discrimination between proteins actively synthesized during the labeling interval from those accumulated at earlier times. Specific procedures for a rapid and convenient method of in vivo pulse labeling are outlined which we believe could be broadly useful in plant biological research. A particularly important technical feature of the protocol is electroblotting of radioactive proteins from the sodium dodecyl sulfate acrylamide resolving gel matrix to polyvinylidene difluoride transfer membrane. This step permits a single polyacrylamide slab gel to be used for autoradiography, immunoblot analysis, and protein staining, thereby greatly facilitating comparison among these complementary techniques and resulting in a significant savings in time and radioactive sample. Central considerations concerning the quantitation and interpretation of in vivo pulse labeling data are discussed.
Martino-Catt
S J
SJ
Photosynthesis Research Unit, Agricultural Research Service/USDA, Urbana, Illinois.
Jones
T L
TL
Ort
D R
DR
D000596:Amino Acids / Q000378:metabolism*; D001345:Autoradiography; D002736:Chloroplasts / Q000378:metabolism; D002940:Circadian Rhythm; D003080:Cold Temperature; D004586:Electrophoresis / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D007553:Isotope Labeling / Q000379:methods*; D008567:Membranes, Artificial; D010940:Plant Proteins / Q000032:analysis* / Q000378:metabolism; D010944:Plants / Q000378:metabolism*; D011145:Polyvinyls; D013194:Staining and Labeling / Q000379:methods; D013462:Sulfur Radioisotopes; D005465:Fluorocarbon Polymers
D000596:Amino Acids; D008567:Membranes, Artificial; D010940:Plant Proteins; D011145:Polyvinyls; D013462:Sulfur Radioisotopes; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers