. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. REPORTS FROM MBL GENERAL SCIENTIFIC MEETINGS 351. 67- 45- Figure 1. LAM was purified as described previously (10). Meihylamine (MA) treatment involved incubation with M methylamine in M Tris buffer, pH for 12 h. at room T. LAM was reacted with bio/in- conjugated trypsin (b-lrypsin) for 2 mm. then boiled in SDS-polyacryl- amide gel electrophoresis sample buffer containing $-mercaptoethanol, and subjected in electrophoresis on a 6% polyacrylamide gel. Western blots were probed with horseradish peroxidase-conjugated
. The Biological bulletin. Biology; Zoology; Biology; Marine Biology. REPORTS FROM MBL GENERAL SCIENTIFIC MEETINGS 351. 67- 45- Figure 1. LAM was purified as described previously (10). Meihylamine (MA) treatment involved incubation with M methylamine in M Tris buffer, pH for 12 h. at room T. LAM was reacted with bio/in- conjugated trypsin (b-lrypsin) for 2 mm. then boiled in SDS-polyacryl- amide gel electrophoresis sample buffer containing $-mercaptoethanol, and subjected in electrophoresis on a 6% polyacrylamide gel. Western blots were probed with horseradish peroxidase-conjugated avidin lo reveal the presence of b-trypsin-containing products. Vnreacted LAM (lane 2) had an apparent molecular mass of 185 kDa, similar lo the molecular mass oj the subunit of human aM (lane 7). The Urn- molecular mass hands in lanes 2 and 7 are thermal breakdown products oj a:M lormed when the samples were boiled in sample bii/ler just be/ore SDS-poly- acrylamide gel electrophoresis. and are thus an artifact of sample prep- aration and do not represent contaminants (11). Trypsin-reacled LAM showed high molecular mass products of apparent molecular masses of ca. 200 and 250 kDa (a 300-350 kDa product was also present in some samples) that migrated slower than the native polypeplide, plus uncom- plexedproteolyticfragments with apparent molecular masses of 100 and 85 kDa that, based on our understanding of the reaction of human a:M with proteases, are presumed to be the result of proteolync cleavage of LAM at the "hull" region (lane 3). Pretreatmenl of LAM with methyl- amine abolished the formation of high molecular mass complexes during the subsequent reaction with trypsin (lane 4). Western blots probed with HRP-avulin lo reveal the complexes with b-trypsin (lanes 5 and 6) showed faintly staining trypsin-containing products with molecular masses ca. 100 and 120 kDa (lane 5) that were absent when methylamme-lreated LAM was reacted with trvpsm (lane 6). Human a:M lorm
Size: 1600px × 1561px
Photo credit: © Library Book Collection / Alamy / Afripics
License: Licensed
Model Released: No
Keywords: ., bookauthorlilliefrankrat, booksubjectbiology, booksubjectzoology
