Pd spintrap g 25 column

Fluorescent dye JF-646 (Grimm et al., 2015 (link)) NHS-ester building block (TOCRIS) was conjugated with Halo-tag ligand amine O4 (Promega) by synthetic chemistry according to published protocols (Grimm et al., 2017 (link)). Briefly, 1.5 equivalents of amine O4 ligand were added to one equivalent of the JF-646 NHS-ester in DMF followed by adding 5% triethylamine. The reaction was vigorously stirred for 16 hr at room temperature and the product was purified by silica gel chromatography, dried by SpeedVac (ThermoFisher), and reconstituted in DMSO.
For optical trapping/confocal microscopy assays, HaloTag Alexa Fluor 488 Ligand (Promega) was utilized as described above, followed by desalting through a PD SpinTrap G-25 column (GE Healthcare) according to the manufacturer’s protocol to remove unreacted dye before use. To label the Halo-tagged actin-binding proteins with Halo-JF-646 for TIRF microscopy assays, two equivalents of synthesized Halo-JF-646 dye was added to the protein solution, followed by incubation for at least 2 hr in the dark at 4°C before use. Subsequent removal of excess dye was not required, as JF-646 is a fluorogenic dye (Grimm et al., 2015 (link)).

Protamine sulfate (2 mg/mL in PBS) was amino-terminally coupled to the bifunctional cross-linker Sulfo-SMCC (2 mg/mL in PBS) at 1:5 molar ratio for 1 h at room temperature on a shaker. PD Spin Trap G25 column (GE Healthcare No.28-9180-04, Chicago, IL, USA) was used to remove uncoupled protamine or SMCC. Conjugates were then mixed with anti-mouse CD19 mAb (32 umol/L, BioXcell, Lebanon, NH, USA) in a 25:1 molar ratio at 4 °C overnight. Non-reacted products and protamine doublets were separated from anti-CD19-SMCC-protamine product using NAP-10 desalting columns (GE Healthcare No.17-0854-01). The anti-CD19-SMCC-protamine conjugates were stored at 4 °C and were stable for several months.

GB1 was expressed in bacteria as a ¹⁵N-labeled protein and purified as described above, then lyophilized and stored at –80 °C. The protein was resuspended and unfolded by incubation in 6 M guanidine hydrochloride containing D₂O to replace the naturally occurring exchanging ¹H protons by ²H. The guanidine hydrochloride was then replaced by PBS buffer made with D₂O (a tablet of Phosphate saline buffer from SIGMA (cat#P4417) was resuspended in 200 ml of D₂O) using a PD SpinTrap G-25 column (GE Healthcare). For the H/D experiments an aliquot of the protein was 1:50 diluted into standard PBS buffer (without D₂O) and measured by NMR after 4 and 12 h of incubation at 37 °C. Transexpression of deuterated GB1 into mammalian A2780 cells was performed and immediately after the protein was analyzed by in-cell NMR at 37 °C in experiments of 4 h and 12 h duration. The same sample was used to record the H/D exchange after 4 and 12 h by NMR. To account for signal loss with time due to signal loss in cells and to normalize for relative transexpressed protein amount when compared to the in vitro control, cross peak intensities of fast exchanging ¹⁵N-¹H moieties identified in the in vitro control experiment (i.e. ¹⁵N-¹H moieties of Glu17, Thr18, and Val23, Figure S3a and S3b) were used.

We followed the RHEB charging protocol as previously described, with some modifications [11 (link)]. RHEB was incubated with 20-fold molar excess of GTPγS (Millipore (Merck), Darmstadt, Germany) in the presence of 10 mM EDTA for 20 min at RT. Finally, the reaction was stopped by the addition of 20 mM of MgCl₂. The yield was then passed over a PD SpinTrap G-25 column (GE Healthcare, Chicago, IL, USA) to remove excess GTPγS.

Aβ–4242 was dissolved in 10 mm NaOH, incubated 10 min on ice and diluted with 50 mm phosphate buffer, pH 7.4, to a final concentration of 40 μm and total volume of 140 μL. In the case of DEPC modification, DEPC was added (30 times excess), the sample was incubated for 30 s or 60 min, and the reaction was stopped with the addition of 10 mm imidazole. All samples (control, 30‐second DEPC‐modified and 60‐min DEPC‐modified Aβ1–42) were applied to 1 mL PD SpinTrap G‐25 Column (GE Healthcare, Chicago, IL, USA) equilibrated with 20 mm 4‐(2‐hydroxyethyl)‐1‐piperazineethanesulfonic acid (HEPES) buffer containing 100 mm NaCl, pH 7.3, and spun down for 1 min at 800 g. The desalted fraction of Aβ1–42 (140 μL) was diluted with 20 mm HEPES buffer containing 100 mm NaCl, pH 7.3, to required volume of 500 μL.

Alexa Fluor 647 NHS Ester (ThermoFisher Scientific) was dissolved in anhydrous-DMSO to a concentration of 2 mg/mL. 5 μL of the dye solution was added to 1 mL 5 mg/mL HI (91077C, Sigma-Aldrich, 95%) monomer solution, mixed gently and thoroughly. The mixed solution was allowed to react for ~2 h at room temperature to complete the conjugation. After that the labeled protein was purified from the excess of free dye by a PD SpinTrap G-25 column (GE Healthcare), divided into aliquots, and stored at −80 °C.
HI spherulites were formed in 0.5 M NaCl, 20% acetic acid (VWR Chemicals, 98%) solution with pH around 1.7. The ratio of labeled to unlabeled HI monomer was about 1–60,000 (dSTORM) or 1–10,000 (REPLOM), with the final concentration of HI was 5 mg/mL. The solution was filtered through 0.22 μm filters (LABSOLUTE) and then incubated in a block heater.