Single-shot cloning of multiple cDNAs coding for a set of related microtubule-associated proteins.
We describe a method for isolating multiple cDNA clones coding for a set of related proteins from bacteriophage lambda expression libraries in a single screening with polyclonal antiserum. The antiserum is raised against a tissue sub-fraction containing the proteins of interest; in the example presented this was brain microtubules. Each antibody-positive clone from the lambda expression library is plaque-purified and then grown in contact with nitrocellulose membrane that becomes coated with protein synthesized from the cloned cDNA. Each filter, containing the protein produced by a single lambda cDNA clone, is then used to affinity-select clone-specific antibodies from the original polyclonal antiserum. The monospecific antibody for each cDNA clone can be used on Western blots to identify the protein that the cDNA encodes and also to stain tissue sections. Using this method we have, in a single screening, obtained: (1) multiple cDNA clones representing different regions of a single large protein, (2) cDNA clones representing several functionally related proteins (microtubule-associated proteins), and (3) cDNA clones related to a novel protein species for which neither biochemical nor immunological data have previously been available.
Garner
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CC
Friedrich Miescher-Institut, Basel, Switzerland.
Farmer
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L
Matus
A
A
D000818:Animals; D000906:Antibodies / Q000302:isolation & purification; D015152:Blotting, Northern; D015153:Blotting, Western; D003001:Cloning, Molecular; D004247:DNA / Q000235:genetics*; D051379:Mice; D008869:Microtubule-Associated Proteins / Q000235:genetics*; D012313:RNA / Q000302:isolation & purification
D000906:Antibodies; D008869:Microtubule-Associated Proteins; D012313:RNA; D004247:DNA