TY - JOUR
T1 - Tubulin isotype specificity and identification epitope for antibody Tub 2.1
AU - Yang, Hailing
AU - Cabral, Fernando
AU - Bhattacharya, Rajat
N1 - Funding Information:
This work was supported by the National Science Foundation (MCB-0516080 to F.C.).
PY - 2009/10
Y1 - 2009/10
N2 - Tub 2.1, a monoclonal antibody commonly used to measure cellular tubulin content, is widely believed to recognize all β-tubulin isotypes. Though it has been used for more than two decades, the epitope for this antibody is not well established. We report for the first time that contrary to common belief, this antibody does not react with all isotypes of β-tubulin. Of the seven vertebrate β- tubulins, the more divergent class V and VI isotypes are not recognized by this antibody. Among the isotypes that do react, binding is similar for (β2, (β3, β4a and β4b but lower for β1, the most abundant isotype. Expression of chimeric tubulins verified that the epitope for Tub 2.1 is near the C-terminal end of β-tubulin. Site-directed mutagenesis of this region in nonreactive β5 to match the sequence of β4b resulted in strong reaction to Tub 2.1 and narrowed the epitope to amino acids 431-436.
AB - Tub 2.1, a monoclonal antibody commonly used to measure cellular tubulin content, is widely believed to recognize all β-tubulin isotypes. Though it has been used for more than two decades, the epitope for this antibody is not well established. We report for the first time that contrary to common belief, this antibody does not react with all isotypes of β-tubulin. Of the seven vertebrate β- tubulins, the more divergent class V and VI isotypes are not recognized by this antibody. Among the isotypes that do react, binding is similar for (β2, (β3, β4a and β4b but lower for β1, the most abundant isotype. Expression of chimeric tubulins verified that the epitope for Tub 2.1 is near the C-terminal end of β-tubulin. Site-directed mutagenesis of this region in nonreactive β5 to match the sequence of β4b resulted in strong reaction to Tub 2.1 and narrowed the epitope to amino acids 431-436.
KW - Multiple tubulin isotypes
KW - Pan specific antibody
KW - Variable reactivity
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U2 - 10.1093/protein/gzp046
DO - 10.1093/protein/gzp046
M3 - Article
C2 - 19625384
AN - SCOPUS:77957684319
SN - 1741-0126
VL - 22
SP - 625
EP - 629
JO - Protein Engineering, Design and Selection
JF - Protein Engineering, Design and Selection
IS - 10
ER -