Fluorescence measurements were performed using a Tecan analyzer infinite 200Pro (Tecan Group Ltd, Männedorf, Switzerland). For mCherry, the excitation wavelength was 587 nm and the emission wavelength was 610 nm. For sfGFP and GFPmut3.1, the excitation wavelength was 485 nm and the emission wavelength was 520 nm. For quantification, calibration was performed using in‐house purified target proteins. We purified mCherry with immobilized metal affinity chromatography using His‐Tag‐specific binding on an Ni‐Sepharose HP column (GE Healthcare Bio‐Sciences, Uppsala, Sweden). GFP and sfGFP were purified through anion exchange chromatography using a Capto Q column (GE Healthcare Bio‐Sciences, Uppsala, Sweden), followed by hydrophobic interaction chromatography using a Butyl Sepharose HP column (GE Healthcare Bio‐Sciences, Uppsala, Sweden), and finally size exclusion chromatography using a Superdex 75 column (GE Healthcare Bio‐Sciences, Uppsala, Sweden). We applied the Beer–Lambert law to determine the concentrations of the standards using the absorbance at 280 nm and the corresponding excitation coefficients.
Capto q column
Manufactured by GE Healthcare
Sourced in Sweden
The Capto Q column is a lab equipment product used for ion exchange chromatography. It is designed to capture and purify target molecules from complex mixtures based on their charge properties. The Capto Q column provides a high-performance separation platform for a variety of biomolecules, including proteins, peptides, and nucleic acids.
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4 protocols using capto q column
1
Fluorescence Quantification Protocol
2
Purification and Characterization of Pvs25-FhCMB
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N. benthamiana plants expressing Pvs25-FhCMB were harvested at 6 dpi and stored at ≤-60 °C. Plants were homogenized, extracted with Triton X-100 (0.5%), centrifuged (16,000 × g, 15 min, 4 °C) and filtrated (Sartopore 2, 0.45/0.2 μm) prior to loading onto IMAC resin (Ni-Sepharose Fast-Flow, GE Healthcare). Proteins was eluted and loaded onto a CaptoQ column (GE Healthcare). Eluted fraction was concentrated 10-fold by centrifugation (Centricon-70, 30 kDa MWCO) and aliquots were frozen at ≤-60 °C. LicKM was expressed as described previously [29] (link), [30] (link).
SDS-PAGE was performed on a 10% acrylamide gel and Coomassie stained. For Western blot, samples were blocked with I-Block (Applied Biosystems). Blots were developed using anti-4xHis mAb (Qiagen). Protein concentration of Pvs25-FhCMB was determined by UV−vis spectrometry using the denaturing method of Edelhoch (1967) with the theoretical extinction coefficient of 73,960 M−1 cm−1. Analytical size exclusion chromatography was performed on a Zenix300 HPLC column (Sepax Technologies, Inc.).
SDS-PAGE was performed on a 10% acrylamide gel and Coomassie stained. For Western blot, samples were blocked with I-Block (Applied Biosystems). Blots were developed using anti-4xHis mAb (Qiagen). Protein concentration of Pvs25-FhCMB was determined by UV−vis spectrometry using the denaturing method of Edelhoch (1967) with the theoretical extinction coefficient of 73,960 M−1 cm−1. Analytical size exclusion chromatography was performed on a Zenix300 HPLC column (Sepax Technologies, Inc.).
3
Recombinant Trx Protein Purification
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A single colony of E. coli BL21 (DE3) was grown overnight in a Luria–Bertani (LB) medium
with 100 μg/mL kanamycin or ampicillin. The overnight culture
was inoculated into a 200 mL LB medium. 1 mM IPTG was added to the
LB medium upon reaching an optical density of 0.6–0.8 at 37
°C, and the culture was grown for an additional 6 h at 30 °C.
The cells were harvested by centrifugation and then resuspended in
buffer A (50 mM Tris·HCl, pH 8.0, 50 mM NaCl, 5 mM imidazole,
and 0.1 mM EDTA), followed by sonication on ice. The supernatant was
loaded into a 1 mL HisTrap column equilibrated with buffer A. The
recombinant Trx proteins were eluted with a linear gradient of 20–300
mM imidazole in buffer A. Proteins in elution fraction were loaded
into a 5 mL Capto Q column on an AKTA go system (GE Healthcare). The
proteins were then eluted with a linear gradient of 50–1000
mM NaCl in buffer B (50 mM Tris·HCl, pH 8.0, and 50 mM NaCl).
All fractions containing the target proteins were pooled. The purified
proteins were kept in buffer B with 5% glycerol and stored at −80
°C for further experiments.
with 100 μg/mL kanamycin or ampicillin. The overnight culture
was inoculated into a 200 mL LB medium. 1 mM IPTG was added to the
LB medium upon reaching an optical density of 0.6–0.8 at 37
°C, and the culture was grown for an additional 6 h at 30 °C.
The cells were harvested by centrifugation and then resuspended in
buffer A (50 mM Tris·HCl, pH 8.0, 50 mM NaCl, 5 mM imidazole,
and 0.1 mM EDTA), followed by sonication on ice. The supernatant was
loaded into a 1 mL HisTrap column equilibrated with buffer A. The
recombinant Trx proteins were eluted with a linear gradient of 20–300
mM imidazole in buffer A. Proteins in elution fraction were loaded
into a 5 mL Capto Q column on an AKTA go system (GE Healthcare). The
proteins were then eluted with a linear gradient of 50–1000
mM NaCl in buffer B (50 mM Tris·HCl, pH 8.0, and 50 mM NaCl).
All fractions containing the target proteins were pooled. The purified
proteins were kept in buffer B with 5% glycerol and stored at −80
°C for further experiments.
4
Recombinant Epsilon Toxin Purification
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The resulting green juice was clarified by filtration through four layers of cheese cloth then loaded onto an equilibrated Capto Q column (GE Healthcare, Piscataway, NJ) with three column volumes of buffer A (50 mM diethanolamine, pH 9.5) and the flow-through/wash fraction collected. The unbound proteins were washed and the bound proteins were eluted. The r-Etx-containing fraction was finally ‘polished’ by Superdex 220-pg size-exclusion chromatograph (Bio-Rad, Hercules, CA) in 50 mM diethanolamine buffer, pH 9.5. The r-Etx was then trypsinated and subsequently formalin inactivated as described (Chandran et al. 2010 (link)) resulting in a protein code named r-Etox, which was then used for subsequent analysis. Residual toxicity tests and formulation methods were also according to Chandran et al. (2010 (link)) with minor modifications. The formalin treatment was conducted to ensure that there was complete processing of the r-Etx into r-Etox as per standard protocols.
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