Chemiluminescence detection of proteins from single cells.
The analysis of proteins from single cells requires techniques of supreme sensitivity. Although radiochemical procedures are capable of detecting small amounts of electrophoretically separated proteins, their sensitivity falls short of that required for routine detection of minor components of single cells. Utilizing the avidin-biotin interaction and the alkaline phosphatase substrate 3-(4-methoxyspiro[1,2-dioxetane-3,2'- tricyclo-[3.3.1.1(3,7)]decan]-4-yl)phenyl phosphate (AMPPD), we have developed an alternative, chemiluminescence-based method for protein detection whose sensitivity exceeds that of other methods. Applying this method to a purified protein, we could detect as little as 63 fg (0.9 amol) of biotinylated bovine serum albumin. The sensitivity of the method was demonstrated by the detection of proteins from individual photoreceptor outer segments, including proteins constituting approximately 1% of the total. Chemiluminescence detection also proved extremely sensitive for immunoblotting: a comparison of five methods for detection of antibody-antigen interactions showed that the AMPPD technique was more sensitive than detection with a colorimetric alkaline phosphatase substrate, 125I-labeled protein A, 125I-labeled anti-mouse IgG, or colloidal gold-conjugated anti-mouse IgG.
Gillespie
P G
PG
Department of Cell Biology and Neuroscience, University of Texas Southwestern Medical Center, Dallas 75235-9039.
Hudspeth
A J
AJ
D000469:Alkaline Phosphatase; D000818:Animals; D001360:Avidin; D001710:Biotin; D002477:Cells / Q000737:chemistry*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D015151:Immunoblotting / Q000379:methods; D007202:Indicators and Reagents; D008163:Luminescent Measurements; D011506:Proteins / Q000032:analysis*; D011892:Rana catesbeiana; D012374:Rod Cell Outer Segment / Q000737:chemistry*; D012710:Serum Albumin, Bovine / Q000032:analysis
D007202:Indicators and Reagents; D011506:Proteins; D001360:Avidin; D012710:Serum Albumin, Bovine; D001710:Biotin; D000469:Alkaline Phosphatase