Premium
Identification of different galectins by immunoblotting after two‐dimensional polyacrylamide gel electrophoresis with immobilized pH gradients
Author(s) -
Lutomski Didier,
Caron Michel,
Cornillot JeanDenis,
Bourin Philippe,
Dupuy Catherine,
Pontet Michel,
Bladier Dominique,
JoubertCaron Raymonde
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170331
Subject(s) - galectin , isoelectric focusing , polyacrylamide gel electrophoresis , gel electrophoresis , lectin , biochemistry , silver stain , chemistry , protein subunit , two dimensional gel electrophoresis , microbiology and biotechnology , chromatography , biology , proteomics , enzyme , gene
Vertebrate soluble β‐galactoside‐binding lectins form a growing protein family that recently have been named galectins. Seven different galectins have been sequenced and characterized in mammals, and there is compelling evidence for the existence of other members of this lectin family. Three among six galectins are homodimers with (i) an identical subunit of a relative molecular mass of about 14500, and (ii) amino acid sequence homologies giving rise to possible immunochemical cross‐reactivities. They are indistinguishable from each other by conventional sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE), even when followed by immunoblottin. However, their different isoelectric points allow their identification using isoelectric focusing and two‐dimensional (2‐D) polyacrylamide gel electrophoresis. A strategy was developed to identify these galectins in crude extracts from cells and tissues, based on the two‐dimensional electrophoresis with immobilized pH gradient (IPG‐Dalt) analysis of the specific spots of purified galectins and of the spots of crude extracts, after silver staining. In addition, 2‐D immunoblotting using anti‐galectin 1 (Gal‐1) and anti carbohydrate‐binding protein 15 (CBP15) antibodies were performed on brain and leukemia cells (HL60) allowing an identification of related polypeptides. Our results indicate that the use of IPG‐Dalt provides a suitable reproducibility and allows the detection of galectins or other galactoside‐binding proteins even at basic p/s.