Hitrap sp hp

Manufactured by Cytiva

Sourced in Sweden

HiTrap SP HP is a strong cation exchange chromatography column designed for the purification of proteins and other biomolecules. It features a porous agarose-based matrix with sulfopropyl (SP) ligands that can effectively bind and separate positively charged species.

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12 protocols using hitrap sp hp

Purification of Bacterial Initiation Factors

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Both initiation factors were purified by Cation exchange chromatography on HiTrap SP HP (Amersham, Uppsala, Sweden). Supernatants were manually loaded to the column (1 mL column volume) and subsequently subjected to a linear NH₄Cl gradient (0.05–1 M) in a Jasco HPLC system (Jasco, Tokyo, Japan). The gradient was prepared in Buffer_A (50 mM Hepes pH 7.1, 10% Glycerol, 6 mM 2-Mercaptoethanol). IF3 and IF1 were eluted at 700 mM and 400 mM of NH₄Cl, respectively, in (Figures S1a and S2a). The best separation conditions were 1 mL/min flow rate and 20 Column Volumes (CV) long gradient, collecting fractions of 1 mL each. Protein elution was followed by absorbance at 290 nm and SDS-Polyacrylamide Gel Electrophoresis (SDS-PAGE, 15%) (Figures S1 and S2). While IF3 was eluted with an elevated degree of purity, IF1 fractions contained high molecular weight contaminates (Figure S2b). Full elimination of the contaminants was obtained by subjecting the combined IF1 fractions to Amicon^® Ultra 30K Da centrifugal filters (Merck) followed by concentration on a HiTrap SP HP (Amersham), single step eluted with 1 M NH₄CL Buffer_A (Figure S2c).
30S subunits purification methods are described in detail in [38 (link)].

Chulluncuy R., Espiche C., Nakamoto J.A., Fabbretti A, & Milón P. (2016). Conformational Response of 30S-bound IF3 to A-Site Binders Streptomycin and Kanamycin. Antibiotics, 5(4), 38.

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Purification of Antifungal Protein from P. chrysogenum

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P. chrysogenum CECT 20922 was grown in potato dextrose broth (PDB; Scharlab, Barcelona, Spain) pH 4.5 incubated statically for 21 days at 25 °C. The mycelium was removed and the medium was filtered through a 0.22 μm-pore-size nylon membrane (MSI, Westboro, USA) to obtain a cell free medium. PgAFP was isolated from 450 mL of the cell-free medium through fast protein liquid chromatography (FPLC) with a cationic exchange column HiTrap SP HP (Amersham Biosciences, Uppsala, Sweden), further purified with a HiLoad 26/60 Superdex 75 gel filtration column (Amersham Biosciences) as previously described [37 (link)]. The isolated and concentrated protein was sterilized through filtration (0.22 µm, Thermo Fisher Scientific, Waltham, MA, USA) and its concentration was assessed by the Lowry method [38 (link)].

Delgado J., Rodríguez A., García A., Núñez F, & Asensio M.A. (2018). Inhibitory Effect of PgAFP and Protective Cultures on Aspergillus parasiticus Growth and Aflatoxins Production on Dry-Fermented Sausage and Cheese. Microorganisms, 6(3), 69.

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Purification and Characterization of PgAFP

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P. alli-sativi CECT 20922, PgAFP producer, was inoculated into malt extract broth (20 g/L malt extract, 20 g/L glucose, and 1 g/L peptone; MEB), pH was adjusted at 4.5, and statically incubated for 21 days at 25 °C. PgAFP was isolated and concentrated from the cell-free medium by fast protein liquid chromatography (FPLC), with a cationic exchange column HiTrap SP HP (Amersham Biosciences, Uppsala, Sweden), further purified with a HiLoad 26/60 Superdex 75 gel filtration column for FPLC (Amersham Biosciences), and concentrated in 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, as previously described (Acosta et al., 2009; (link)Rodríguez-Martín et al., 2010) (link).
A pooled stock solution of PgAFP was measured by the Lowry method (Lowry et al., 1951) , sterilised through 0.22 μm acetate cellulose filters (Fisher Scientific, United Kingdom), and stored at -20 °C until use.

Delgado J., Ballester A.R., González-Candelas L, & Núñez F. (2022). Impact of the antifungal protein PgAFP on the proteome and patulin production of Penicillium expansum on apple-based medium. International journal of food microbiology, 363.

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Purification and Characterization of PgAFP

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The PgAFP-producing P. chrysogenum CECT 20922 was inoculated into Malt Extract Broth (MEB, 20 g/L malt extract, 20 g/L glucose, and 1 g/L peptone; MEB) at pH 4.5, and incubated for 21 days at 25 °C. PgAFP protein was obtained from the cell free medium by fast protein liquid chromatography (FPLC) with a cationic exchange column HiTrap SP HP (Amersham Biosciences, Sweden) and further purified and desalted through a HiLoad 26/60 Superdex 75 gel filtration column for FPLC (Amersham Biosciences, Sweden) (Acosta et al., 2009) (link).
The concentration of the pooled PgAFP stock solution was measured by the Lowry method (Lowry et al., 1951) , sterilised through 0.22 μm acetate cellulose filters (Jet Bio-Filtration Co., China), and stored at -20 °C until use.

Delgado J., Núñez F., Asensio M.A, & Owens R.A. (2019). Quantitative proteomic profiling of ochratoxin A repression in Penicillium nordicum by protective cultures. International journal of food microbiology, 305.

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Purification of Antifungal Protein from P. chrysogenum

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The PgAFP-producing P. chrysogenum CECT 20922 was inoculated into malt extract broth (20 g/L malt extract, 20 g/L glucose, and 1 g/L peptone; MEB), pH was adjusted at 4.5, and incubated up to 21 days at 25 °C. PgAFP was obtained from the cell-free medium by fast protein liquid chromatography with a cationic exchange column HiTrap SP HP (Amersham Biosciences, Uppsala, Sweden), further purified with a HiLoad 26/60 Superdex 75 gel filtration column for FPLC (Amersham Biosciences), and concentrated in 50 mM sodium phosphate buffer (pH 7.0) containing 0.15 M NaCl, as previously described (Acosta et al., 2009; Rodríguez-Martín et al., 2010) (link).
PgAFP concentration in a pooled stock solution was measured by Lowry method (Lowry et al., 1951) , sterilized through 0.22 µm acetate cellulose filters (Fisher Scientific, United Kingdom), and stored at -20 °C until use.

Delgado J., Ballester A.R., Núñez F, & González-Candelas L. (2019). Evaluation of the activity of the antifungal PgAFP protein and its producer mould against Penicillium spp postharvest pathogens of citrus and pome fruits. Food microbiology, 84.

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Purification of Antifungal Protein PgAFP

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P. chrysogenum was grown in potato dextrose broth (PDB; Scharlab, Barcelona, Spain) pH 4.5, at 25 °C for 21 days. Mycelium was removed and the culture medium was filtered through a nitrocellulose 0.22-μm pore size (Sartorius, Goettingen, Germany) to obtain cell-free medium. The cellfree medium was applied to an ÄKTA FPLC with a cationic exchange column HiTrap SP HP (Amersham Biosciences, Uppsala, Sweden), equipped with a UV detector at 214 nm. The resulting fraction containing PgAFP was then chromatographed on a HiLoad 26/60 Superdex 75 column for FPLC (Amersham Biosciences, Uppsala, Sweden) as previously described (Acosta et al. 2009) (link) to obtain an extract of the purified protein. The extract containing the purified PgAFP from several batches were pooled in a stock solution. The amount of protein in the stock solution was quantified by the Lowry method (Lowry et al. 1951) . For the various assays, the PgAFP stock solution was diluted to the desired active concentration range according to the aim of the test.

Delgado J., Owens R.A., Doyle S., Asensio M.A, & Núñez F. (2015). Impact of the antifungal protein PgAFP from Penicillium chrysogenum on the protein profile in Aspergillus flavus. Applied microbiology and biotechnology, 99(20).

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Recombinant Expression and Purification of rVAR2 Protein

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All recombinant rVAR2 proteins were expressed in E. coli Shuffle cells (NEB) using a pET28 vector. The constructs of rVAR2 (ID1-ID2a) contained either a SpyTag [22 (link),23 (link)] or an albumin-binding domain (ABD) [24 (link)] for extension of plasma half-life fused in the N-terminal, and in the C-terminal a V5-tag and 6×His-tag were added for detection and affinity purification. The control proteins, DBL4 and DBL5, both contained a V5-tag and His-tag in the C-terminal similar to rVAR2 and for DBL5 an ABD was fused in the N-terminal.
Expression and purification of the rVAR2 protein were performed, as described in a previous publication [16 (link)]. Briefly, the E. coli pellet harboring the recombinant protein was resuspended in lysis buffer with benzonase and homogenized. After centrifugation and sterile-filtration, the supernatant was loaded onto a HisTrap HP (Cytiva, Uppsala, Sweden) column and eluted using imidazole. The protein was further purified by HiTrap SP HP (Cytiva, Uppsala, Sweden), before elution an endotoxin reduction wash step was included where 0.1% Triton X114 in binding buffer was passed over the column. After removing the wash buffer, protein was eluted with a linear gradient from 0 to 1 M NaCl in 25 mM phosphate buffer pH 7.2. The eluted rVAR2 protein was pooled and analyzed by SDS-PAGE, aliquoted, flash-frozen, and stored at −80 °C until use.

Oo H.Z., Lohinai Z., Khazamipour N., Lo J., Kumar G., Pihl J., Adomat H., Nabavi N., Behmanesh H., Zhai B., Dagil R., Choudhary S., Gustavsson T., Clausen T.M., Esko J.D., Allen J.W., Thompson M.A., Tran N.L., Moldvay J., Dome B., Salanti A., Al-Nakouzi N., Weiss G.J, & Daugaard M. (2021). Oncofetal Chondroitin Sulfate Is a Highly Expressed Therapeutic Target in Non-Small Cell Lung Cancer. Cancers, 13(17), 4489.

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Purification of PDZ Tandem-NZ-1 Fab Complex

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Using the plasmids constructed above, the PA14-inserted PDZ tandem fragments were produced as N-terminal glutathione S-transferase (GST)-fusion proteins, in which a TEV protease recognition site was incorporated between the GST and PDZ tandem sequences, as reported previously (Hizukuri et al., 2014 ▸ ). Each PA-inserted construct was overproduced in E. coli BL21(DE3) cells and purified from the cell lysate using Glutathione Sepharose 4B resin (Cytiva). The mutant fragment was cleaved from the GST portion through on-column digestion with TEV protease, and the released fragment, which contained two additional residues (Gly-Ser) upstream of the PDZ tandem, was further purified using cation-exchange chromatography (HiTrap SP HP, Cytiva) and size-exclusion chromatography (Superdex 200 Increase 10/300 GL, Cytiva). In parallel, the NZ-1 Fab was prepared by cleaving the NZ-1 antibody using papain and purifying as reported previously (Fujii et al., 2016 ▸ ). NZ-1 was obtained from the Antibody Bank (http://www.med-tohoku-antibody.com/topics/antibody.htm) at Tohoku University, Miyagi, Japan. The purified PDZ tandem mutant was mixed with the NZ-1 Fab in a 2:1 molar ratio and was applied to size-exclusion chromatography to fractionate the complex. The final protein sample was concentrated by ultrafiltration.

Tamura-Sakaguchi R., Aruga R., Hirose M., Ekimoto T., Miyake T., Hizukuri Y., Oi R., Kaneko M.K., Kato Y., Akiyama Y., Ikeguchi M., Iwasaki K, & Nogi T. (2021). Moving toward generalizable NZ-1 labeling for 3D structure determination with optimized epitope-tag insertion. Acta Crystallographica. Section D, Structural Biology, 77(Pt 5), 645-662.

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Purification and Characterization of Acidaminococcus sp. dCas12a

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The Acidaminococcus sp dCas12a protein was expressed in Escherichia coli BL21 and purified by chromatography on Ni-NTA (Cytiva), HiTrap SP HP (Cytiva), and HiLoad Superdex 200 16/60 (Cytiva) columns and determined purity through polyacrylamide gel electrophoresis. The 55 nt guide RNA (gRNA) was transcribed in vitro and then purified by TRIzol (Invitrogen) and verified integrity through denaturing urea polyacrylamide gel electrophoresis.

Torres-García E., Pinto-Cámara R., Linares A., Martínez D., Abonza V., Brito-Alarcón E., Calcines-Cruz C., Valdés-Galindo G., Torres D., Jabloñski M., Torres-Martínez H.H., Martínez J.L., Hernández H.O., Ocelotl-Oviedo J.P., Garcés Y., Barchi M., D’Antuono R., Bošković A., Dubrovsky J.G., Darszon A., Buffone M.G., Morales R.R., Rendon-Mancha J.M., Wood C.D., Hernández-García A., Krapf D., Crevenna Á.H, & Guerrero A. (2022). Extending resolution within a single imaging frame. Nature Communications, 13, 7452.

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Purification and Interaction of mDPPA3 Fragment

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The cDNA encoding mDPPA3 (residues 61-150) was cloned into the modiﬁed pET21b vector, pET-N^pro vector (39 (link)). The N^pro-fused mDPPA3 was puriﬁed from the pellet fraction. The inclusion body was solubilized and denatured in buffer containing 8 M urea, 50 mM Tris-HCl [pH 7.5], 25 mM DTT by stirring overnight at 4°C. The denatured protein was puriﬁed by Ni Sepharose 6 Fast Flow (Cytiva) and. The eluents were dialyzed in a step-wise manner to gradually remove the urea. The solution was incubated with a buffer containing 50 mM Tris-HCl [pH 8.5], 200 mM NaCl, 2 mM DTT for 24 h at room temperature for complete autocleavage of N^pro. The mDPPA3_61-150 was further puriﬁed using HiTrap SP HP and HiLoad 16/60 Superdex 30 (Cytiva). MagneGST™ Glutathione Particles (Promega) were used for the assay. The truncated GST-mUHRF1 (10 μg) were immobilized on the beads (10 μl) equilibrated with the binding buffer (20 mM Tris–HCl [pH 7.5], 150 mM NaCl, 1 mM DTT, 10% glycerol and 0.5% NP-40). After the unbound proteins were washed-out thrice by the 200 μl of the binding buffer, 10 μg of C-terminal fragment of mDPPA3 (residues 61–150 or 76–128 W) was incubated with the beads for 2 h at 4°C. After incubation, the unbound protein was washed thrice using 200 μl of the binding buffer. The bound proteins were boiled for 2 min at 95°C in an SDS sample buffer and analyzed by SDS-PAGE.

Hata K., Kobayashi N., Sugimura K., Qin W., Haxholli D., Chiba Y., Yoshimi S., Hayashi G., Onoda H., Ikegami T., Mulholland C.B., Nishiyama A., Nakanishi M., Leonhardt H., Konuma T, & Arita K. (2022). Structural basis for the unique multifaceted interaction of DPPA3 with the UHRF1 PHD finger. Nucleic Acids Research, 50(21), 12527-12542.

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