Pd 10 gel filtration column

PTD-ODD-Luciferase (POL), ODD-Luciferase (OL) and PTD-ODDmutant-Luciferase (POmL) fusion proteins were expressed in BL21-CodonPlus cells (Stratagene, La Jolla, CA, USA) as GST-tagged fusion proteins. The GST-tagged proteins were prepared essentially as described previously18 (link) and purified with a GST-column and digested with precision protease (GE Healthcare, Waukesha, WI, USA) to remove GST-tag from the fusion protein. The final products were equilibrated in Mg/Ca-free PBS (pH = 7.4). The fusion proteins (20 nmol/mL) were mixed with Alexa Fluor 680 C-2 maleimide (60 nmol/60 μL) (Invitrogen, Carlsbad, CA, USA) at 4 °C for 12 h. POL-AF probes were purified with a PD-10 gel filtration column (GE Healthcare) and an Amicon-10 centrifugation column (Millipore, Milford, MA, USA). The purified POL-AF was finally resolved in PBS (pH 7.4). Fluorescence-labeling characterizations were confirmed by SDS-PAGE fluorescence imaging and the labeling rate was calculated by colorimetric method with dye dilution series. The labeling rate was >0.9.

The mouse monoclonal antibody ATN-291 (1.5 mg in 290 µL PBS) was reacted with CHX-A”-DTPA-p-Bn-SCN (5 µL, 20 μmol/mL in DMSO, Macrocyclics) after pH adjustment to 9 with aq. sodium carbonate (50 µL, 100 mM). After 1 h of gentle agitation in a 37 °C water bath the solution was passed over a PD-10 gel filtration column (GE Life Sciences) in 100 mM pH 5 sodium acetate, and the large molecule fraction was collected in 1.5 mL of effluent. Efficacy of the conjugation was confirmed through test radiolabeling with ¹⁷⁷Lu.
100 μL of the ATN-291 solution (protein concentration: 1 μg/μL) was added to a vial containing 500 μL of the radionuclide solutions (0.1 M HCl) and 150 μL 1 M ammonium acetate. The solutions were incubated at 37 °C for 90 min, and again for 60 min after addition of aq. EDTA (100 μL, 50 mM, pH 7) to scavenge unchelated radionuclides, and the radiolabeling efficiency was checked with Al-backed silica TLC plates run in 1:1 acetonitrile and 0.5 M sodium acetate (pH 5). The solutions were then loaded onto a fresh PD-10 column, prepped and eluted with HEPES buffered (10 mM, pH 7.4) isotonic saline. 1.5 mL of eluate, containing the radiolabeled antibodies, were collected for injection.

Extraction and purification of Flamindo/Flamindo2 protein and in vitro spectroscopy were performed as previously reported [20] (link). Briefly, a Flamindo/Flamindo2-containing pRSET_B vector was introduced into Escherichia coli JM109 (DE3) cells and cells were cultured at 20°C for 4 days. After 4 days of culture, cells were harvested by centrifugation, lysed by three freeze-thaw cycles, and sonicated with lysozyme. After centrifugation of the lysate, His-tagged Flamindo/Flamindo2 protein was purified from supernatants using a Ni-NTA agarose column (Qiagen) followed by a PD-10 gel-filtration column (GE Healthcare). The purified protein was finally eluted in Hepes buffer (150 mM KCl and 50 mM Hepes-KOH [pH 7.4]). The concentration of purified Flamindo/Flamindo2 protein was measured by the Bradford protein assay using Bio-Rad Protein Assay (Bio-Rad). Bovine serum albumin was used as a standard. Absorption and fluorescence spectra of purified Flamindo/Flamindo2 protein were measured using a UV-670 UV-Vis spectrophotometer (Jasco) and F-2700 fluorescence spectrophotometer (Hitachi), respectively.

Human galectins were prepared as outlined previously. The recombinant galectin-3 was purified by affinity chromatography on lactosyl-Sepharose, and the bound lectin was eluted with 100mM lactose in PBS and 14mM β-mercaptoethanol (β-ME). Prior to derivatization, β-ME was removed from the galectin-3 samples by passing through a PD-10 gel filtration column (GE Healthcare), followed by the addition of 100mM lactose to help maintain the stability of galectin-3 and reduce the likelihood of adduct formation at or near the carbohydrate recognition domain. Alexa Fluor 488-labeled galectin-3 was prepared with either Alexa Fluor 488 C5-maleimide or Alexa Fluor 488 carboxylic acid succinimidyl ester dilithium salt-reactive dyes (Molecular Probes) according to manufacturer’s protocol.

Purified proteins C4BP or Protein H (40 μg) were labeled with each 18.5 MBq (Hartmann Analytics) in the presence of 2 iodobeads (Pierce, Thermo Fisher) for 15 min according to the manufacturer's instruction. Free radioactivity was removed using a PD-10 gel filtration column (GE Healthcare) and eluted with 500 μl PBS. Fractions containing the majority of radioactivity (usually 3) were pooled and used for experiments. For storage at −20°C 500 μl glycerol was added. Specific activity was measured by analyzing 2 μl of the glycerol stock in a Wizard² gamma counter (Perkin Elmer). ¹²⁵I-C4BP had an average specific activity of about 30.000 cpm/μl, while ¹²⁵I-Protein H had in average 75.000 cpm/μl.