Abstract
A procedure for the molecular identification of MHC class I products based on 1-D IEF and subsequent immunoblotting is described. Optimal conditions for 1-D IEF, the electrophoretic transfer of proteins out of denaturing, nonionic detergent-containing gels to nitrocellulose, and the requisite antibodies, both polyclonal and monoclonal, for the visualization of class I heavy chains have been established. Cross-reactivity of antibodies has enabled the biochemical analysis of class I heavy chains in the dog. The procedure reported here requires modest amounts of cells and allows a rapid molecular characterization of class I heavy chain polymorphisms in man and other species without the need for radiochemical methods.
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Abbreviations
- FCS:
-
fetal calf serum
- MHC:
-
major histocompatibility complex
- NP-40:
-
Nonidet P-40
- PBL:
-
peripheral blood lymphocytes
- PHA:
-
phytohemagglutinin
- RaHC:
-
rabbit anti-heavy chain serum
- TX-114:
-
Triton X-114
- 1-D IEF:
-
one-dimensional isoelectric focusing
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Neefjes, J.J., Doxiadis, I., Stam, N.J. et al. An analysis of class I antigens of man and other species by one-dimensional IEF and immunoblotting. Immunogenetics 23, 164–171 (1986). https://doi.org/10.1007/BF00373817
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DOI: https://doi.org/10.1007/BF00373817