Detection of the hypusine-containing protein (HP = eIF-5A) in crude yeast extracts by two-dimensional western blots.
The hypusine-containing protein (HP) with its unique modification of a specific lysine residue resulting in the amino acid hypusine is highly conserved among all eukaryotes and is also found in Archaebacteria. Studies of the protein function in translational processes showed a stimulatory effect in the methionyl puromycin assay, but not in in vitro translation of native mRNA. It was therefore also designated as eIF-5A. To further investigate the role of HP in cellular metabolism, we purified the protein from Saccharomyces cerevisiae and raised polyclonal antibodies in chicken. Immunoglobulin preparations from the eggs of the immunized hens were used for Western blot analysis of HP in crude yeast extracts. For those studies, the soluble protein fraction of the yeast was resolved on two-dimensional gels (first dimension: isoelectric focusing using an immobilized pH gradient (IPG), pH 4-7; second dimension: sodium dodecyl sulfate-polyacrylamide gel electrophoresis, 12% T) and subsequently blotted onto Fluorotrans membrane. Anodic versus cathodic application of the extracts of the IPG strips was compared.
D015153:Blotting, Western / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000302:isolation & purification*; D008239:Lysine / Q000031:analogs & derivatives / Q000032:analysis; D010448:Peptide Initiation Factors / Q000302:isolation & purification*; D016601:RNA-Binding Proteins; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D012834:Silver; D013194:Staining and Labeling; D000097574:Eukaryotic Translation Initiation Factor 5A
D005656:Fungal Proteins; D008239:Lysine; D010448:Peptide Initiation Factors; D016601:RNA-Binding Proteins; D012834:Silver; D000097574:Eukaryotic Translation Initiation Factor 5A; C100028:hypusine