Abstract
Generation of specific antibodies against enriched subcellular fractions is a powerful strategy to identify and characterize cellular components. We show that recombinant antibodies can be selected in vitro by phage display against complex subcellular fractions, namely microtubule-binding proteins and Golgi stacks. This technique has allowed us to overcome many limitations of the classical animal-based approach and generate cell biology-compliant antibodies. In addition, we show that intracellular expression of GFP-tagged recombinant antibodies can reveal the dynamics of endogenous proteins in vivo. Endogenous Giantin is very static and outlines the Golgi in living cells. It accumulates neither onto Golgi-derived tubules upon Brefeldin A treatment before Golgi disappearance, nor onto de novo formed Golgi mini-stacks upon microtubule depolymerization, and remains instead on the 'old' pericentriolar Golgi. This suggests that, in contrast to other Golgi matrix proteins, endogenous Giantin is very stably associated with the Golgi and does not efficiently recycle to the ER. Altogether, we show that the antibody phage display technique represents an efficient alternative to rapidly generate versatile antibodies that represent new tools to study protein function.
Publication types
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Research Support, Non-U.S. Gov't
MeSH terms
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Animals
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Biomarkers
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Brefeldin A / pharmacology
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Golgi Apparatus / chemistry*
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Golgi Apparatus / drug effects
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Golgi Apparatus / immunology
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Golgi Apparatus / metabolism*
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Golgi Matrix Proteins
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Green Fluorescent Proteins
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HeLa Cells
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Humans
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Immunoglobulin Variable Region / immunology
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Immunoglobulin Variable Region / metabolism*
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Luminescent Proteins / immunology
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Luminescent Proteins / metabolism*
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Membrane Proteins / genetics
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Membrane Proteins / metabolism
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Microtubule-Associated Proteins / immunology
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Microtubule-Associated Proteins / isolation & purification
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Microtubule-Associated Proteins / metabolism
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Microtubules / metabolism
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Peptide Library
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Protein Synthesis Inhibitors / pharmacology
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RNA, Small Interfering / metabolism
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Rats
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Recombinant Fusion Proteins / genetics
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Recombinant Fusion Proteins / immunology
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Recombinant Fusion Proteins / metabolism*
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Single-Chain Antibodies
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Subcellular Fractions / chemistry*
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Subcellular Fractions / immunology
Substances
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Biomarkers
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Golgi Matrix Proteins
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Immunoglobulin Variable Region
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Luminescent Proteins
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Membrane Proteins
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Microtubule-Associated Proteins
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Peptide Library
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Protein Synthesis Inhibitors
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RNA, Small Interfering
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Recombinant Fusion Proteins
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Single-Chain Antibodies
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macrogolgin
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scFV green fluorescent protein, recombinant
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Green Fluorescent Proteins
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Brefeldin A