Comparative sensitivity of /sup 125/I-protein A and enzyme-conjugated antibodies for detection of immunoblotted proteins
Immunoblotting is a powerful technique for the detection of small amounts of immunologically interesting proteins in unpurified preparations. Iodinated protein A (PA) has been widely used as a second antibody for detection of proteins; however, it does not bind equally well to immunoglobulins from different species nor does it bind to all subclasses of immunoglobulin G (IgG). We compared the sensitivity of (/sup 125/I)PA with those of both horseradish peroxidase-conjugated second antibodies (HRP) and glucose oxidase-anti-glucose oxidase (GAG) soluble complexes for visualizing bovine serum albumin, human IgG, or human C3 which was either dot blotted or electroblotted to nitrocellulose. (/sup 125/I)PA was uniformly 10- to 100-fold less sensitive than either HRP or GAG. GAG was more sensitive than HRP except for C3 (electroblotting) and bovine serum albumin and IgG (dot blotting), in which they were equivalent. In general, dot blotting was 10- to 1000-fold more sensitive than electroblotting. Although relative sensitivities varied depending on the proteins analyzed and the antisera used, GAG appeared to be superior to (/sup 125/I)PA and HRP for detection of immunoblotted proteins.
- Research Organization:
- Huntington Veterans Administration Medical Center, Huntington, WV
- OSTI ID:
- 6859984
- Journal Information:
- J. Clin. Microbiol.; (United States), Journal Name: J. Clin. Microbiol.; (United States) Vol. 1; ISSN JCMID
- Country of Publication:
- United States
- Language:
- English
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59 BASIC BIOLOGICAL SCIENCES
ANIMALS
ANTIBODIES
BETA DECAY RADIOISOTOPES
CATTLE
COMPARATIVE EVALUATIONS
DAYS LIVING RADIOISOTOPES
DOMESTIC ANIMALS
ELECTRON CAPTURE RADIOISOTOPES
ENZYMES
GLOBULINS
IMMUNOGLOBULINS
IMMUNOLOGY
INTERMEDIATE MASS NUCLEI
IODINE 125
IODINE ISOTOPES
ISOTOPES
MAMMALS
MAN
NUCLEI
ODD-EVEN NUCLEI
ORGANIC COMPOUNDS
OXIDOREDUCTASES
PEROXIDASES
PRIMATES
PROTEINS
RADIOISOTOPES
RUMINANTS
SENSITIVITY
VERTEBRATES