74 



A. A. KANDUTSCH AND JACK H. STIMPFLING 



(B) Extraction of antigenic activity from the particulate fraction. 

 In a previous publication, extraction of antigenic activity by a 

 5 per cent solution of Triton, pH 9, at room temperature, was 

 reported (Kandutsch, i960). We have since found that the yield 



WASHED SA. I (ASCITES) CELLS 



Homogenize in dist. H20 



Centrifuge -discord supernatant froction. 



SEDIMENT 



Homogenize sn dist. H20 



Digest l.5hrs.at ZVC with DNose (5mg/l00ml), 



RNase(12mg/IOOml) a.025M MgCh. 

 Centrifuge -discard supernatant fraction. 



SEDIMENT 



Wash I X with 0.14 M NaCI and at least 3x with 



dist. HaO. 

 Freeze -dry. 



PARTICULATE FRACTION 



Homogenize I gm in 40ml 5% Triton. Add a crystal 

 of Thymol and allow to stond 10-14 days at 

 0-4''C maintaining pH at 7.5 with Tris. 

 Centrifuge at 105,000 xg for I hr. -discard sediment. 

 SUPERNATANT 



Pour into 10 vols. Acetone (- 20° C ) , 

 Centrifuge -wash 2 x with ether. 



SEDIMENT (Whole Triton extract) 



I 



SEDIMENT 



Extract 2x with 0.1 M phosphate pH 7.2, 4x with 

 O.I4MNaCI. 



SUPERNATANT FRACTION 

 ( Water Soluble Fraction of Triton Extract) 



(Triton Soluble Lipoprotein 

 TSL ) 



Fig. I. Isolation of Triton-soluble lipoprotein (TSL) from Sarcoma i 



(ascites form). Unless otherwise indicated, centrifugation was for 30 



minutes at 60,000 g. 



of antigenic activity in the extract is much higher when extraction 

 is carried out for from lo to 14 days at refrigerator temperatures 

 at a pH maintained at about 7* 5 by the addition of tris (tris(hydro- 

 xymethyl)aminomethane). After the extraction period the 

 mixture is centrifuged at 105,000 g for one hour. Material is 

 recovered, free of Triton, from the supernatant fraction by 

 precipitation with ten volumes of cold acetone followed by an 



