A nonradioactive biochemical characterization of membrane proteins using enhanced chemiluminescence.
Here we demonstrate a nonradioactive immunoprecipitation and sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) technique which replaces the standard practice of isotopic protein labeling by iodination or metabolic tagging in the analysis of membrane proteins. The technique has proved extremely valuable in the biochemical analysis of small quantities of frozen, pathological tissue. Membranes were prepared from Dx3 (a human melanoma cell line), C6 (a rat glial cell line), and osteoclastoma (a human giant cell tumor of bone). The membranes were labeled with biotin and immunoprecipitated with a variety of antibodies to the vitronectin receptor (VNR). The VNR proteins were resolved by SDS-PAGE and immunoblotted onto nitrocellulose paper. The biotinylated protein was visualized using streptavidin horseradish peroxidase and enhanced chemiluminescence (ECL). Film exposures ranged from 15 min to 16 h. Good visualization of the VNR, yielding the typical heterodimeric receptor of 90 and 150 kDa, was given. Signals generated were high and background noise low with a 30-min film exposure. An overnight exposure increased the detection of weaker bands. In conclusion, biotinylation of membrane proteins proved a satisfactory label for immunoprecipitation and SDS-PAGE analysis. The ECL development stage was extremely flexible with visualization of strong and weak signals. The method has several advantages over a conventional radioactive immunoprecipitation in that it is relatively inexpensive, simple, quick and nonhazardous.
Nesbitt
S A
SA
Imperial Cancer Research Fund, St. Bartholomew's Hospital, London, United Kingdom.
Horton
M A
MA
D001710:Biotin; D015153:Blotting, Western / Q000379:methods; D002460:Cell Line; D002462:Cell Membrane / Q000737:chemistry* / Q000648:ultrastructure; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005870:Giant Cell Tumors; D006801:Humans; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D008545:Melanoma; D008565:Membrane Proteins / Q000032:analysis* / Q000302:isolation & purification; D008970:Molecular Weight; D010010:Osteoclasts / Q000737:chemistry; D014407:Tumor Cells, Cultured
D007202:Indicators and Reagents; D008565:Membrane Proteins; D001710:Biotin