A low level of interaction between full-length 140Q-htt and 3xFlag20Q-htt but not 3xFlag 7Q-htt can be detected by co-immunoprecipitation. (A) 700 μg of cytoplasmic protein prepared from the striatum (Str) and cerebellum (Cb) of a 6 month-old Hdh 140Q/3xFlag7Q mouse was immunoprecipitated with anti-Flag agarose beads, and separated into antibody non-bound (NB) and antibody-bound (B) fractions. Duplicate samples (Input Str represents 56 μg protein, while each NB sample represents 8% of the fraction, and each B sample represents 10% of the fraction for the anti-FLAG blot, and 40% of the fraction for the 1C2 blot) were fractionated on 5% SDS-PAGE and analyzed by western blotting with the indicated antibodies. 140Q-htt was not detected in the B-fractions, suggesting that a stable interaction between 140Q-htt and 3xFlag7Q-htt was below detection limits. (B) 500 μg whole brain cytoplasmic extract from a 13 month-old Hdh 3xFlag140Q/HA7Q mouse was immunoprecipitated with anti-FLAG or 1C2 antibodies using Protein G-agarose beads, and the NB- and B-fractions were analyzed by western blotting using 1C2 and 2166 antibodies. (C) 500 μg whole brain cytoplasmic extract from 27 month-old Hdh 140Q/3xFlag7Q and 16 month-old Hdh 140Q/3xFlag20Q mice was immunoprecipitated with anti-FLAG antibody and Protein-G agarose beads, and the NB- and B-fractions were analyzed by western blotting using 1C2 and 2166 antibodies. Shorter and longer exposures of the western blots are shown in the top two panels of (B,C). As a negative control for non-specific binding of htt to the agarose beads, antibody was omitted from the mixture of protein extract and Protein G-agarose beads (No Ab) in (B,C). An arrowhead marks the position of 140Q-htt co-immunoprecipitating with 3xFlag20Q-htt, while an asterisk indicates 3xFlag20Q-htt that is detected inefficiently by the 1C2 antibody when it is present in large amounts (C). The position of a 250 kD protein standard is indicated on the left.