Pd 10 desalting column

To determine whether the peroxidase activity is cysteine-dependent, rPbPrx1 (2 mg mL⁻¹) was treated with DTT for 1 h at room temperature. The excess DTT was removed by gel filtration using a PD-10 desalting column (GE Healthcare, Piscataway, USA). The reduced protein was incubated in 1 mM N-ethylmaleimide (NEM) (Sigma, München, Germany) overnight at 4°C. The excess NEM was removed by gel filtration using a PD-10 desalting column (GE Healthcare, Piscataway, USA). N-ethylmaleimide (NEM) is an alkylating reagent that reacts with sulfhydryl groups, thus blocking the Prx activity. The reactions were performed at 30°C in a solution containing 50 mM Hepes-NaOH (pH 7.4), 100 μM DTPA, 1 mM sodium azide, 12.5 μM rPbPrx1, 10 mM DTT, and 5 mM t-BOOH. The peroxidase activity was monitored by the DTT oxidation (λ = 310 nm).

Ubiquitin G76C mutant was reduced with 5 mM 2-mercaptoethanol (Nacalai Tesque) and then buffer-exchanged into 50 mM sodium phosphate buffer pH 7.5 using a PD-10 desalting column (GE Healthcare). The reduced ubiquitin mutant was mixed with a 20-fold molar excess of 5,5′-dithiobis-(2-nitrobenzoic acid) (DTNB, Tokyo Chemical Industry) and then incubated for ~2 hours at room temperature with linear shaking. The reaction solution was buffer exchanged into ligation buffer (20 mM Tris-HCl, 50 mM NaCl and 1 mM EDTA, pH 7.0) using a PD-10 desalting column. The reduced ubiquitin mutant was mixed with a 2-fold molar excess of the DTNB-activated ubiquitin mutant in which a disulfide bond had been established between the thiol groups of DTNB and the C-terminal cysteine of the ubiquitin G76C mutant; subsequently, this solution was incubated for 1 hour at room temperature with linear shaking. After residual DTNB was removed using a PD-10 desalting column, the resultant disulfide-conjugated diubiquitin (Ub₂^S-S) was separated from DTNB-activated and/or reduced ubiquitin mutants in 50 mM sodium phosphate and 150 mM NaCl, pH 7.0 by using a Hiload 16/60 Superdex 75 pg size exclusion column (GE Healthcare).

The [¹H-¹⁵N]–TROSY HSQC NMR spectra of AnFld WT and the 2A mutant were recorded using Watergate solvent suppression at 25 °C on a Bruker 600 MHz spectrometer equipped with the (¹H/¹⁹F)-X broadband CryoProbe Prodigy. [U-¹⁵N]-labeled proteins were exchanged into NMR sample buffer (50 mM potassium phosphate buffer (pH 7.5) containing 0.1 M NaCl) using a PD10 desalting column (GE Healthcare). The samples were 0.2 mM of protein dissolved in a mixture of 90% (v/v) NMR sample buffer (in ¹H₂O) and 10% (v/v) ²H₂O. The spectra were acquired using a total of 2048 complex points in t2 and 512 increments in t1 with 8 scans per increment over a spectral width of 9.6 and 2.1 kHz in the ¹H and ¹⁵N dimensions, respectively. To study the interaction of AnFld and Spy, [U-²H, ¹⁵N, ¹³C]-labeled AnFld and Spy were exchanged into 50 mM potassium phosphate buffer (pH 7.5) without any salt using a PD10 desalting column (GE Healthcare). ¹⁵N-TROSY spectra were collected at 25 °C with 0.2 mM AnFld dissolved in 90% (v/v) buffer (in ¹H₂O) and 10% (v/v) D₂O and then titrations were done by addition of appropriate volume of Spy to the NMR tube. A total of 2048 data points in t2 dimension and 256 increments in t1 with 16 scans per increment were acquired. The spectra were processed using NMRPipe suite, analyzed, and plotted in NMRFAM Sparky^{53 (link),54 (link)}.