Detection and quantification of biotinylated proteins using the Storm 840 Optical Scanner.
The use of the avidin-biotin interaction is becoming an increasingly common method for the detection of proteins. The use of fluorescence detection with avidin-biotin systems has the potential to greatly increase both the sensitivity and linearity of this type of analysis. In this report, three fluorescent systems were tested for their ability to detect biotinylated polypeptides in purified and complex biological samples. These systems include a Neutravidin-Alexa Fluor430 conjugate, an avidin-horseradish peroxidase conjugate with the ECL-Plus detection system, and an avidin-alkaline phosphatase conjugate with the ECF detection system. Biotinylated molecular weight standards, biotinylated bovine serum albumin, and rat liver homogenate were resolved by SDS-PAGE gel electrophoresis and transferred to polyvinyldifluoride membrane. Biotinylated polypeptides were then visualized on the Storm840 optical scanner. The Neutravidin-Alexa Fluor430 conjugate exhibited the lowest sensitivity, but displayed high linearity. The avidin-horseradish peroxidase and avidin-alkaline phosphatase conjugates, when combined with appropriate fluorescent substrates, exhibited much higher fluorescence, with the avidin-alkaline phosphatase ECF system displaying the highest sensitivity. All systems demonstrated an ability to reliably detect and quantify biotinylated polypeptides in purified as well as complex samples, given careful attention to conditions optimized for each system.
Lewis
Brandon
B
Food Science and Human Nutrition Department, Institute of Food and Agricultural Sciences and the, College of Agricultural and Life Sciences, University of Florida, Gainesville, FL 32611-0370, USA.
Rathman
Sara
S
McMahon
Robert J
RJ
D000103:Acetyl-CoA Carboxylase / Q000032:analysis; D000469:Alkaline Phosphatase; D000818:Animals; D001360:Avidin; D019921:Biotinylation; D019735:Carbon-Carbon Ligases / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D006735:Horseradish Peroxidase; D007527:Isoenzymes / Q000032:analysis; D008099:Liver / Q000737:chemistry / Q000201:enzymology; D008297:Male; D043586:Methylmalonyl-CoA Decarboxylase / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D011766:Pyruvate Carboxylase / Q000032:analysis; D051381:Rats; D017207:Rats, Sprague-Dawley; D012680:Sensitivity and Specificity; D012710:Serum Albumin, Bovine / Q000032:analysis / Q000737:chemistry; D013050:Spectrometry, Fluorescence / Q000295:instrumentation / Q000379:methods*
D007527:Isoenzymes; D011506:Proteins; D001360:Avidin; D012710:Serum Albumin, Bovine; D006735:Horseradish Peroxidase; D000469:Alkaline Phosphatase; D019735:Carbon-Carbon Ligases; D011766:Pyruvate Carboxylase; D000103:Acetyl-CoA Carboxylase; C021573:methylcrotonoyl-CoA carboxylase; D043586:Methylmalonyl-CoA Decarboxylase