User:Anthony Salvagno/Notebook/Research/2010/07/05/Tethering Research
- Details about the flow chambers for TPM measurements have been reported.16,17 A flow chamber was incubated overnight at 4 °C with 40 μg/mL biotin-labeled BSA (Sigma, St. Louis, MO). After washing the flow chamber with 800 μL of buffer (10 mM Tris−HCl (pH 7.4), 200 mM KCl, 5% dimethyl sulfoxide (DMSO), 0.1 mM ethylenediaminetetraacetic acid (EDTA), 0.2 mM dithiothreitol (DTT), and 0.1 mg/mL α-casein), the chamber was incubated for 2 h with 50 μg/mL streptavidin (Sigma, St. Louis, MO). Separately, DNA was incubated for 2 h with an excess of antidigoxigenin-coated beads (480 nm in diameter, Indicia Diagnostics, Oullins, France) and suspended in buffer lacking DTT and DMSO. After washing the chamber with 800 μL of buffer, biotin- and digoxigenin-labeled DNA was introduced and incubated for 1 h. Unattached beads were then flushed from the chamber with buffer.
So they do a lot of incubating. Their setup seems like it is opposite ours (biotin on glass with anti-dig beads) but they use the .5um beads and incubate overnight.
- Unless otherwise specified, all chemicals and reagents were purchased from Sigma–Aldrich. The sample was prepared as follows. All steps were performed at room temperature; long incubations were conducted in a water-saturated container to avoid evaporation of the sample. A solution containing 20 µg/ml anti-digoxigenin (1333089, Roche, IN) in phosphate-buffered saline (PBS) buffer [2.7 mM KCl, 137 mM NaCl, 5.4 mM Na2HPO4 and 1.8 mM KH2PO4 (pH 7.4)] was introduced into the flow chamber and incubated for 20 min to insure optimal surface coating. The excess antibody was then removed by washing the chamber 5–7 times with 80 µl of LBB buffer [10 mM Tris–HCl (pH 7.4), 200 mM KCl, 0.1 mM EDTA, 5% (v/v) dimethyl sulfoxide (DMSO), 0.2 mM DTT, 0.1 mg/ml -casein]. The DNA sample (80 µl at a concentration of 20 ng/ml in LBB buffer: this DNA concentration was chosen to maximize single DNA tethers as described below) was then introduced in the chamber and incubated for 1 h. Unbound DNA was removed by washing with 7 x 50 µl LBB– buffer (LBB lacking DTT and DMSO). Streptavidin-coated polystyrene microspheres (diameter 440 nm, Indicia Biotechnology, France) in LBB– buffer were then introduced in the flow chamber and incubated for 30 min. Unbound microspheres were finally removed with 5 x 50 µl washes of LBB buffer supplemented with Lac repressor at a tetramer concentration of 4, 20 or 100 pM, depending on the experiment. The flow chamber was then sealed with silicon grease to allow prolonged observation at the microscope (each slide could be observed for several hours without detectable loss in activity of LacI).
Interesting. Their protocol is very similar to mine but with longer incubation times. They also use 0.44um beads for their DNA tethering. This protocol is with 1.5kb PCR tethers.
- A flow chamber (with a volume of 20 µl) was constructed between a microscope slide and a coverslip kept at a distance of 60 µm by double-sided tape. Strips of silicon grease were deposited on the inner side of the tape to provide lateral sealing and avoid contact of the solution with the tape itself. The microscope slides were previously cleaned in an ultrasonic bath in ethanol for 5 min. The coverslips were cleaned further by a 10 min treatment with reactive ion plasma in a radio-frequency plasma cleaner (PDC-002, Harrick Inc., NY).
Same flow chamber but they seal the tape with silicon grease. I will have to try the cleaning method for the glass (minus the plasma cleaner).
- The flow chambers for TPM measurements were similar to those described by Finzi and Dunlap . Aflowchamberwas incubated over night at 4◦Cwith biotin-labelled bovine serum albumin (BSA) (40 μg ml−1; Sigma-Aldrich, Inc., St Louis, MO, USA). After washing with 800 μl of buffer (10 mM Tris-HCl pH 7.4, 200 mM KCl, 5% dimethyl sulphoxide (DMSO), 0.1 mM EDTA, 0.2 mM dithiothreitol (DTT) and 0.1 mgml−1α-casein), the chamber was incubated for 2 h with streptavidin (50 μg ml−1; Sigma-Aldrich). After washing the flow chamber with 800 μl of buffer, DNA in the pM range was introduced into the chamber and incubated for 1 h. This DNA, labelled at one end with biotin and at the other with digoxigenin was pre-incubated for 2 h with an excess of antidigoxigenin-coated beads (430 nm in diameter, Indicia Diagnostics, Oullins, France) in buffer lacking DTT and DMSO. Finally, the unbound DNA and unattached beads were flushed from the chamber with buffer.
Similar setup to that first paper.
I feel like I've read this before but worth rereading.
- Experiments were performed using a protocol described in Pouget et al. (19). A coverslip flow chamber (30 µl vol) was incubated with the anti-digoxygenin antibody (20 mg/ml; Roche) in phosphate-buffered saline (PBS) for 20 min at 4°C. After washing, the chamber was incubated at 4°C overnight in reaction buffer [20 mM HEPES (pH 7.5), 100 mM KCl, 10 mM MgCl2, 2% glycerol, 2 mM DTT, 10% dimethyl sulfoxide (DMSO), 20 µg/ml BSA, 1 mg/ml dephosphorylated -casein (Sigma)]. DNA substrates were incubated in the flow chamber for 1 h and labelled with latex beads (0.2 µm diameter neutravidin-labelled microspheres; Molecular Probes). For the majority of experiments, OrfAB was added by flushing the flow chamber with 100 nM OrfAB. For real observations of synapse assembly, 1–2 µl of 6 µM OrfAB were added at one corner of the chamber to let the protein diffused. The tethered beads were visualized by differential interference contrast (DIC) or fluorescence videomicroscopy at a recording rate of 25 frame/s. Bead position was determined on successive images as the spot centroid using a home built image analysis programme (19).
This one is way different.
This is the reference in the above paper.
- The chambers (50 µl volume) were built by mounting coverslips on microscope slides with double-faced adhesive tape. They were first treated with 20 µg/ml polyclonal anti-digoxigenin antibody (Roche) in PBS for 20 min at 4°C. To reduce non-specific DNA binding to the glass, the chambers were then incubated with the buffer containing dephosphorylated -casein for 1 h at 4°C. A further incubation of 1 h at room temperature in buffer with DNA (3.4 x 10–11 M) modified at one end with digoxigenin and at the other with biotin was followed by extensive washing with buffer. Finally, the DNA molecules attached by their digoxigenin ends were coupled to 0.2 µm diameter neutravidin-labeled microspheres (Molecular Probes) at a final concentration of 0.003% (v/v) by incubation for 20 min at room temperature.
This seems to be the most similar protocol to what I do, but the incubation times are super long. I will try this technique first with added salt for the bigger beads.