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HPLC Isolation of putative sea urchin cell adhesion molecules
Author(s) -
Kiaei Pani,
Kivman Lisa,
Asuncion Frank,
Gaytan Maria,
Alvarez Maribel,
Badali Oliver,
Esmaili Linda,
Nnoli Jennifer,
CoyleThompson Cathy,
Oppenheimer Steven B.
Publication year - 2006
Publication title -
the faseb journal
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.709
H-Index - 277
eISSN - 1530-6860
pISSN - 0892-6638
DOI - 10.1096/fasebj.20.4.a227-b
Subject(s) - sea urchin , high performance liquid chromatography , chromatography , chemistry , embryo , biology , biochemistry , microbiology and biotechnology
Using HPLC anion‐exchange chromatography with a Bio Rad UNO Q1 column, proteins that caused exogastrulation in Lytechinus pictus sea urchin embryos were isolated from desalted calcium‐magnesium‐free sea water disaggregation supernatant. Exogastrulation is the process in which the advancing tip of the archenteron fails to adhere to the roof of the blastocoel and everts out of the embryo proper. The disaggregation supernatant was obtained by dissociating 24–32 hr old L. pictus embryos and fractionated on the UNO Q1 column. The fractions and controls with sea water only were added in quadruplicate to live sea urchin early gastrula embryos in a 96 well microplate, with about 10 embryos per well, incubated at 15 C for 24 hrs and observed live and after fixation with 10% formaldehyde. Two HPLC fractions (8/9 and 10/11) contained statistically significant exogastrulation inducing activity based on 4 replicates (p less than 0.05), compared with controls in sea water. Fraction 8/9 contained a single Coomassie brilliant blue‐staining 35 kD band and fraction 10/11 contained 2 bands (35 kD and 150 kD) on Invitrogen pre‐cast NuPAGE 4%–12% gradient Bis‐TRIS gels. Other labs have isolated similar molecular weight proteins from different sea urchin species and stages. Our ability to obtain large quantities of the purified proteins and to quantitatively assay their effects on well defined adhesive interactions, may help elucidate the nature of molecular mechanisms involved in specific cellular interactions in a model system (supported by NIH NIGMS SCORE, RISE, MARC, ITQ program and the Joseph Drown Foundation).