The calibration curve in microbead sized particles was prepared (Fig. 1a ) using resin similar to Benzamidine Sepharose 4 Fast Flow® but without any pABA linked to it: Immobilized metal ion-affinity chromatography Sepharose Gel (GE Healthcare IMAC Sepharose® 6 Fast Flow). Standard solutions for glyoxal reaction (amidine concentration in the range of 0.05 – 1.00 mM) were prepared as described in section 2.2.2. Fifty μL of the IMAC gel was pipetted in separate wells of a 96-well microplate and 50.0 μL standard glyoxal reaction solution (0.05 – 1.00 mM amidine) added to each well resulting in Sepharose gel containing different amounts of the fluorophore AAIO. The excitation/emission maxima were determined for the AAIO fluorophore in this phase and finally fluorescence in each well measured using excitation and emission wavelengths of 330 nm and 510 nm, respectively (Table 1 and ESM Fig. S2 ).
Imac sepharose 6 fast flow
Manufactured by GE Healthcare
Sourced in United States
IMAC SepharoseTM 6 Fast Flow is a chromatography resin designed for the purification of histidine-tagged recombinant proteins. It consists of a high flow agarose matrix with immobilized metal ion affinity chromatography (IMAC) ligands. The resin enables rapid and efficient capture of target proteins from crude samples.
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Lab products found in correlation
Ktaprime plus system, by GE Healthcare (2 mentions)
Image studio v3, by LI COR (2 mentions)
Human fxa, by Enzyme Research (1 mentions)
Heparan sulfate from bovine kidney, by Merck Group (1 mentions)
Human thrombin, by Merck Group (1 mentions)
Human leukocyte cathepsin g, by Merck Group (1 mentions)
Trypsin, by Merck Group (1 mentions)
Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions)
Human plasmin, by Merck Group (1 mentions)
α chymotrypsin from bovine pancreas, by Merck Group (1 mentions)
Emulsiflex c5 high pressure homogenizer, by Avestin (1 mentions)
5 protocols using imac sepharose 6 fast flow
1
Calibration Curve for Microbead-based IMAC
2
Characterization of Lamprey AGTR1 Receptor
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Human thrombin (≥2 800 NIH U/ml), human plasmin, human leukocyte cathepsin G, trypsin and α-chymotrypsin from bovine pancreas, affinity-isolated anti-hemagglutinin (HA) antibodies from rabbits and heparan sulfate from bovine kidney were purchased from Sigma-Aldrich. Human FXa (176 IU/mg) was from Enzyme Research Laboratories. Horse anti-mouse IgG and Q5 high-fidelity DNA polymerase (2 000 U/ml) were from New England BioLabs. Horseradish peroxidase-linked anti-rabbit antibodies from donkey and IMAC Sepharose 6 Fast Flow were purchased from GE Healthcare. The FXa substrate S-2222 was from Chromogenix. PCR primers (Table S1
3
IMAC Purification of Redox-Sensitive Proteins
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IMAC is a good purification technique for proteins and peptides that exhibits the advantages of high selectivity, high binding capacity and high recovery [25 (link)]. The RSHs were purified according to the method of Lv et al. (2009) [41 (link)] with some modifications. A column (16 mm × 30 cm) was packed with IMAC-Sepharose TM6 Fast Flow (GE Healthcare, Waukesha, WI, USA) and charged with Fe2+ (5 bed volumes, 200 mM FeCl2 · 4H2O). RSHs (20 mg/mL) was filtered through filter membrane (0.22 µm). The nonspecific bound iron was removed with milli-Q water (5 bed volumes), subsequently, 2 mL of the sample solution was loaded onto the column. RSHs were isolated through a two-step elution program. Peptides without affinity to immobilized iron in the column were eluted with distilled water. Then, the bound peptides were eluted using 20 mM phosphate-buffered saline, pH 7.4 (containing 500 mM NaCl), with a flow rate of 2 mL/min. The absorbance of eluates was monitored at 220 nm with UV detector. The elution fractions were collected at room temperature and lyophilized, then stored at −20 °C for further XO inhibitory activity evaluation.
4
Qm1 Protein Purification from SHuffle T7 Express E. coli
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For the Qm1 purification from SHuffle® T7 Express E. coli, the cells were resuspended in PBS buffer and lysed by a mechanical disruption in an EmulsiFlex C-5 High-pressure homogenizer from Avestin (Ottawa, ON, Canada) and centrifuged at 17,500× g for 10 min at 4 °C to separate the pellet of the soluble fraction. The soluble fraction was loaded onto a Ni2+ charged column IMAC SepharoseTM 6 Fast Flow, connected to an ÄKTA prime plus system, both from GE Healthcare (Chicago, IL, USA). The column was equilibrated with five column volumes (CVs) of PBS buffer, then the sample was loaded and the flow-through was collected for analysis. The elution was made using Imidazole in PBS buffer at increasing concentrations: 25, 50, 150, and 400 mM (Supplementary Material, Figure S3 ). The purified Qm1 protein was quantified by SDS-PAGE by densitometry using bovine serum albumin as an external standard. The images were captured and processed using the LI-COR Odyssey imaging system program Image Studio v 3.1 (LI-COR, USA).
5
Purification of Soluble epIL-18 by IMAC
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Soluble epIL-18 was purified by immobilized metal affinity chromatography (IMAC). The soluble fraction from E. coli rupture was loaded into a Ni2+ charged matrix IMAC SepharoseTM 6 Fast Flow (GE Healthcare, Chicago, IL, USA), connected to the GE ÄKTA prime plus system. The decreasing urea gradient (8 to 0 M) was completed to recover not-denaturing conditions. Elution was performed with increasing imidazole concentration (25, 250, and 500 mM). Purified epIL-18 was quantified by SDS-PAGE using bovine serum albumin as an external standard and the recovery percentage of the initial fraction was calculated by densitometry. Images were captured and processed using an LI-COR imaging system program (Image Studio v 3.1, LI-COR, Lincoln, NE, USA).
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