Identification of the B1 and B2 subunits of human placental laminin and rat parietal-yolk-sac laminin using antisera specific for murine laminin-beta-galactosidase fusion proteins

J C Brown, J H Spragg, G N Wheeler, P W Taylor

Research output: Contribution to journalArticlepeer-review

14 Citations (Scopus)

Abstract

Antisera raised against fusion proteins consisting of murine laminin B1 and B2 subunit sequences fused to the C-terminus of Escherichia coli beta-galactosidase were tested for their subunit specificity on Western blots of deglycosylated murine Engelbreth-Holm-Swarm (EHS) laminin. The antisera raised against B2 subunit sequences (anti-XLB2.1 and anti-XLB2.2) bound only to the EHS laminin B2 subunit. One of the antisera raised against B1 subunit sequences (anti-XLB1.2) was specific for the B1 subunit, whereas two others (anti-XLB1.1 and anti-XLB1.3) cross-reacted with the EHS laminin B2 subunit. Gold-labelled heparin-albumin was shown to bind specifically to the A subunit of deglycosylated EHS laminin on Western blots. These reagents were used to identify the homologous subunits in rat parietal-yolk-sac laminin and human placental laminin. The anti-(fusion protein) antisera identified the B1 and B2 subunits of the rat laminin, and these were similar in size to the murine EHS B subunits. Human placental laminin gave bands of 400, 340, 230, 190 and 180 kDa on reducing SDS/PAGE. The anti-(fusion protein) antisera identified the 230 and 190 kDa bands as the B1 and B2 subunits respectively. Gold-labelled heparin-albumin bound to the 400, 340 and 190 kDa bands of human placental laminin and so did not unambiguously identify a single A subunit. The human placental laminin may contain a mixture of isoforms, with alternative subunits substituting for the A subunit.
Original languageEnglish
Pages (from-to)463-468
Number of pages6
JournalBiochemical Journal
Volume270
Issue number2
DOIs
Publication statusPublished - 1 Sep 1990

Keywords

  • Amino Acid Sequence
  • Animals
  • Antibody Specificity
  • Western Blotting
  • Polyacrylamide Gel Electrophoresis
  • Escherichia coli
  • Galactosidases
  • Glycosylation
  • Humans
  • Immune Sera
  • Laminin
  • Mice
  • Molecular Sequence Data
  • Molecular Weight
  • Placenta
  • Rats
  • Recombinant Fusion Proteins
  • Nucleic Acid Sequence Homology
  • Yolk Sac
  • beta-Galactosidase

Cite this