Autoradiographic Analysis on Agar Plates of Antigens From Sub Cellular Fractions of Rat Liver Slices

Abstract

Slices of rat livers were incubated with 14C amino acids, homogenized, and subjected to differential centrifugation. The microsomes were further extracted with the non-ionic detergent Lubrol W and with EDTA. These extracts and the microsome free "cell sap," freed from the pH 5 precipitable fraction, were subsequently reacted with antisera using agar diffusion techniques. The antisera employed were obtained from rabbits injected with different subcellular fractions of rat liver or with rat serum proteins. When the agar diffusion plates were autoradiographed it was found that some of the precipitates were radioactive while others were not. Control experiments indicated that this labeling was due to the specific incorporation of 14C amino acids into various rat liver antigens during incubation of the slices rather than to a non-specific adsorption of radioactive material to the immunological precipitates. When the slices were incubated with the isotope for up to 30 minutes, the serum proteins which could be extracted from the microsomes with the detergent were strongly labeled, as were a number of additional microsomal antigens of unknown significance. In contrast, the serum proteins present in the cell sap were only weakly labeled. Most of the typical cell sap proteins, both those precipitable and those soluble at pH 5, seemed to remain unlabeled. No consistently reproducible results were obtained with the EDTA extracts of the ribosomal residues remaining after extraction of the microsomes with the detergent. Incubation of the liver slices for longer periods (up to 120 minutes) led to a strong labeling of the serum proteins in the cell sap as well as to the appearance of labeling in additional cell sap proteins. The results are discussed with regard to the subcellular site of synthesis and the metabolism of the different antigens.

Characterization of the Pattern of Amino Acid Incorporation in Cell-Free Liver Systems by Autoradiography of Precipitin Reactions

“…also 3, 15). With regard to the microsomal extracts, the pattern of incorporation appears, at least roughly, similar to that found after incubation of liver slices (13). With regard to the pH 5 soluble fraction of the cell sap the situation is different.…”

IMMUNOLOGICAL STUDY ON PROGRESSIVE APPEARANCE OF MICROSOMAL CONSTITUENTS OF THE DEVELOPING CHICKEN LIVERS1)2)

“…But it is quite within the bounds of possibility that some of the 'common components' found in microsoma1 fraction may be freshly synthesized molecules which should subsequently be transferred into unsedimented fraction (PERLMANN, HuL'rIN, D'AMELIO and MORGAN, 1959;MORGAN, PERLMANN and HULTIN, 1960). In this respect, the differential distribution of esterase components in different microsomal extracts is particularly interesting.…”

Preparation of projection-less particles from influenza virus and their messenger activities in prokaryotic and eukaryotic systems

“…N e u r a m i n i d a s e i n h i b i t i o n t e s t was carried o u t as described (6). F o r t h e localization of n e u r a m i n i d a s e precipitin lines, w a s h e d i m m u n o d i f f u s i o n plates (29) were e q u i l i b r a t e d with 0.05 • p h o s p h a t e buffer, p i t 6.0, a n d i n c u b a t e d for 10--15 m i n u t e s (37 ° C) w i t h a n overlay of M P N (0.6 mg/ml) a n d F a s t B l a c k K salt (0.5 mg/mt) in t h e same buffer. D i s t i n c t red lines a p p e a r e d w i t h i n 5 minutes.…”

“…T h e n it was m a d e to 1 per cent sodium dodeeyl sulfate (SDS) a n d centrifuged for 10 m i n u t e s a t 15,000 × g. The s u p e r n a t a n t was dialyzed against 0.05 N s o d i u m p h o s p h a t e buffer p H 7.4, e o n t M n i n g 0.85 p e r cent NaCI a n d 1 msI dithiothreitot, t h e n centrifuged for 10 minutes at 15,000 × g. (In some e x p e r i m e n t s t h e SDS t r e a t m e n t was replaced b y t r e a t m e n t w i t h 7 ~x u r e a a n d 1 per cent T r i t o n X-100.) I m m u n o d i f f u s i o n in agar gel a n d s u b s e q u e n t a u t o r a d i o g r a p h i e analysis of labelled p r o t e i n antigens were p e r f o r m e d as described previously (29). I m m u n o e t e c t r o p h o r e s i s was carried o u t as described (51).…”

“…0.85 per cent NaC1. After the precipitation lines had developed, the gels were washed as described (29), followed by rewashing with 1 per cent Triton X-100. Left: Photograph of gel stained with amido black.…”