Deae sepharose fast flow anion exchange column

Human cTnI and cTnT were overexpressed in E. coli strain BL21 (DE3) cells, and protein purification and quantification were performed according to our previous studies [22 (link),26 (link)]. The purified cTnI was labeled with TAMRA, and purified cTnT was labeled with Alexa Fluor 488, respectively. To achieve 1:1 label ratio of both proteins, an optimized protocol was developed based on manufacture suggestion (Thermo Fisher Scientific). Two-fold fluorescent dye was mixed with a known amount of protein sample at pH 8.6 for 30 min at room temperature. Then, the mixture was passed a DEAE Sepharose Fast Flow anion exchange column (GE Healthcare) to fractionate unlabeled, single-labeled, and multiple-labeled proteins [48 (link)]. Under our optimized labeling condition, only the single-labeled fraction was observed and eluted out. After extensive dialysis (4 changes of dialysis buffer) against PBS to remove free fluorescent probes, Absorption measurements were performed to determine the labeling ratio. The molar extinction coefficients of 65,000 cm⁻¹M⁻¹ at 555 nm and 73,000 cm⁻¹M⁻¹ at 495 nm were used to calculate the ratio of TAMRA and Alex Fluor 488 to the known amount cTnI and cTnT, respectively. The overall label ratio is 1:1 for both proteins.

Bacterial cells were collected and sonicated in 50 mM Tris-HCl and 100 mM NaCl buffer, pH 7.4, containing protease inhibitor (Roche, Germany), and bacterial cell debris was removed by centrifugation (12,000g). The supernatant was subjected to a Diethylaminoethyl (DEAE)-Sepharose Fast Flow anion exchange column (GE Healthcare Bio-Sciences AB, Sweden) preequilibrated with 50 mM Tris-HCl pH 7.4 (buffer A) with a KNAUER Azura FPLC system coupled to a UV detector (Germany). The protein sample was loaded onto the column at a flow rate of 0.5 ml/min. The column was washed with buffer A (3 column volumes) at a flow rate of 1 ml/min. Then, a linear gradient was established with buffer A and buffer B (1 M NaCl), and the rest of the bound proteins were eluted by increasing the percentage of buffer B from 0% to 100% during 100 minutes. The UV wavelength was set at 280 nm.