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Phage library panning against cytosolic fraction of cells using quantitative dot blotting assay: application of selected VH to histochemistry
- Source :
- Journal of Immunological Methods. 261:65-72
- Publication Year :
- 2002
- Publisher :
- Elsevier BV, 2002.
-
Abstract
- Comprehensive preparations of antibodies against various kinds of proteins in cells would be useful in proteome research and antibody-based research. Here we report the panning of a human antibody heavy chain variable domain (VH) phage library against a cytosolic fraction of rat liver to obtain antibodies specific for certain cytoplasmic proteins. Rat liver specimens were homogenized and subjected to differential centrifugation. A 125000 x g supernatant (rat liver cytosol, RLC) was immobilized onto a nitrocellulose membrane and subjected to phage VH library panning. For efficient assessment of binding phages, we established a system that was a combination of monoclonal phage ELISA and quantitative dot blotting of phages. The VH genes of the binding phages were selected and expressed as VH--bacterial alkaline phosphatase (PhoA) conjugates (VH/RLC--PhoAs) in Escherichia coli. One of the VH/RLC--PhoAs stained one major band on Western blotting of RLC and also stained the cytoplasm of hepatocytes histochemically. This is the first report of phage library panning against the cytosolic fraction of cells to obtain human VH fragments, and the application of those human VH fragments to histochemical study.
- Subjects :
- Male
Phage display
Blotting, Western
Immunoblotting
Immunology
Immunoglobulin Variable Region
Enzyme-Linked Immunosorbent Assay
Bacteriophage
Cytosol
Peptide Library
Animals
Humans
Immunology and Allergy
Amino Acid Sequence
Rats, Wistar
Peptide library
Panning (camera)
Differential centrifugation
Base Sequence
biology
DNA
biology.organism_classification
Molecular biology
Rats
Blot
Liver
Cytoplasm
biology.protein
Antibody
Immunoglobulin Heavy Chains
Subjects
Details
- ISSN :
- 00221759
- Volume :
- 261
- Database :
- OpenAIRE
- Journal :
- Journal of Immunological Methods
- Accession number :
- edsair.doi.dedup.....41e321898d8fcf92adb92eb713f0b7d0