A Novel Complex: A Quantum Dot Conjugated to an Active T7 RNA Polymerase
Figure 4
Gel filtration of QD : T7RNAP complexes. This experiment was performed to exclude the possibility that the active Bio-T7 RNAP was bound to streptavidin (and not to QDs). (a) Control without QDs using 2.137 nmol Bio-T7 RNAP, (b) control without QDs using 0.385 nmol Bio-T7 RNAP, and (c) 0.385 nmol Bio-T7 RNAP + 0.075 nmol QDs (Bio-T7RNAP : QD 5 : 1). All three graphs show the absorption of 280 nm light as function of time (left axis) as well as the conductivity (right axis). In all three experiments the sample was filtered through a Superose 6 10/300 GL column at a flow rate of 0.5 mL/minute, and fractions of 0.5 mL were collected from 11 to 40 minutes. In each of panels (a), (b), and (c) an inserted gel shows the activity found in the individual fractions. Fraction 32 of panel a is a positive control of Bio-T7 RNAP activity. The DNA MW marker used (left lane in all three inserted gels) was from top 3 kbp, 2 kbp, 1.5 kbp, 1.2 kbp, 1 kbp, 900 bp 800 bp, 700 bp, 600 bp, and so forth. Equal amounts of the DNA MW marker were loaded on each gel. Bio-T7 RNAP (103 kDa) was eluted at 33.2 minutes (fraction 24) and QDs at 24 minutes (fraction 15). In a (not shown) calibration E. coli GlyS (224 kDa) eluted at 28.4 minutes, Dextran blue at 14.5 minutes (fraction 5), and 70 S ribosomes (~3000 kDa) at 20.8 minutes (fraction 13).
