Peaks 1–5 , corresponding to the five TTR tetramers resulting from subunit
exchange, were collected via UPLC after passage through a fluorescence
detector. The peak from plasma detected 1–3 min after injection,
termed peak0 , was also collected. Because the concentration
of TTR in the collected fractions was predicted to be very low, each
peak was collected from four injections of the same sample, pooled,
and concentrated using an Amicon Ultra-4 Centrifugal Filter device
with a 10 kDa cutoff. The concentrated samples were then volume normalized,
analyzed by SDS–PAGE and Western blotting, and detected and
quantified using a Li-Cor Odyssey infrared imaging system. Untagged
TTR was detected using a polyclonal rabbit anti-TTR antibody (DAKO)
at a 1:5000 dilution, and FT2·WT TTR was detected
using the M2 mouse anti-FLAG antibody (Agilent) at a 1:1000 dilution.
exchange, were collected via UPLC after passage through a fluorescence
detector. The peak from plasma detected 1–3 min after injection,
termed peak
of TTR in the collected fractions was predicted to be very low, each
peak was collected from four injections of the same sample, pooled,
and concentrated using an Amicon Ultra-4 Centrifugal Filter device
with a 10 kDa cutoff. The concentrated samples were then volume normalized,
analyzed by SDS–PAGE and Western blotting, and detected and
quantified using a Li-Cor Odyssey infrared imaging system. Untagged
TTR was detected using a polyclonal rabbit anti-TTR antibody (DAKO)
at a 1:5000 dilution, and FT2·WT TTR was detected
using the M2 mouse anti-FLAG antibody (Agilent) at a 1:1000 dilution.