IMMUNOCHEMICAL STUDIES OF THE PLANT PROTEINS 42 1 
caused the production of only a small precipitate, so the whole was 
poured into three volumes of a mixture of acetone and ether (80-20) 
and the precipitate so formed was centrifugalized out and washed in 
alcohol and ether and dried over sulphuric acid under diminished 
pressure. When desiccation was complete it formed a gray powder 
which was more or less insoluble according to the time of exposure to 
the alcohol baths. 
Proteins Insoluble in Water or Neutral Aqueous Saline 
Solutions 
Gliadin and glutenin occur miostly in the endosperm of the seed 
and are the proteins which make up gluten, the substance which gives 
what flour its capacity for making dough. In order to obtain the 
gluten the flour is mixed with water and kneaded into a stiff dough. 
This is then wrapped in muslin and kneaded under water until the 
starch is washed out. When it is mostly removed the dough may be 
taken from the muslin and the kneading continued under water until 
no further starch can be removed. In preparing the proteins for this 
work the gluten was next kneaded in several baths of 10 percent NaCl, 
then chopped fine and allowed to remain in a large volume of salt 
solution over night to complete the removal of the globulin, then 
(still chopped fine) it was allowed to remain in running water for some 
hours to remove the salt. 
In order to dissolve out the gliadin it was now subjected to ex- 
traction with alcohol. This was done by boiling in 70 percent alcohol 
on the hot water bath for about an hour, using a reflux condenser to 
keep the alcohol from evaporating. It was then strained off through 
muslin and the extraction of the undissolved gluten was continued 
with a fresh bath of alcohol. In the meantime the first extract was 
filtered and the alcohol distilled off by heating in a retort on a boiling 
water bath. The alcohol recovered from this distillation was then 
diluted with water to make again 70 percent by the hydrometer test, 
and used for the next bath. This process was repeated five or six 
times or until nearly all the alcohol soluble protein was removed from 
the gluten. Care was taken during the evaporation of the gliadin 
solution in the still not to let too much alcohol evaporate. No 
attempts were made to see what percentage of alcohol remained or at 
what tempen ture it was boiling. Distillation was discontinued, 
however, while there was still enough alcohol left to lower the boiling 
