Isolation of cDNA clones encoding proteins of complex structures: analysis of the Trypanosoma brucei cytoskeleton.
We have adapted a group of well-known procedures in order to devise a simple method that allows the isolation of specific cDNAs encoding proteins located in different regions of the Trypanosoma brucei cytoskeleton. cDNA clones were isolated by screening a lambda gt11 expression library with a polyspecific, polyclonal antiserum against a complex immunogen, in this case the complete cytoskeleton. The fusion proteins produced by the clones were then used as an affinity immunoadsorbant to select monospecific polyclonals. The monospecific antisera isolated were used as probes to identify and localize different cytoskeleton proteins by Western blotting and immunofluorescence. This method proved particularly useful for the molecular identification of minor components in a complex structure. It should prove applicable to the molecular analysis of other organelles or protein complexes.
Birkett
C R
CR
Biological Laboratory, University of Kent, Canterbury, U.K.
Parma
A E
AE
Gerke-Bonet
R
R
Woodward
R
R
Gull
K
K
D000818:Animals; D000913:Antibodies, Protozoan / Q000737:chemistry; D000918:Antibody Specificity; D003001:Cloning, Molecular; D003599:Cytoskeleton / Q000737:chemistry*; D016054:DNA, Protozoan / Q000302:isolation & purification*; D007106:Immune Sera / Q000737:chemistry; D046911:Macromolecular Substances; D015800:Protozoan Proteins / Q000235:genetics* / Q000302:isolation & purification; D011817:Rabbits; D014346:Trypanosoma brucei brucei / Q000737:chemistry / Q000235:genetics*
D000913:Antibodies, Protozoan; D016054:DNA, Protozoan; D007106:Immune Sera; D046911:Macromolecular Substances; D015800:Protozoan Proteins