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Hitrap sp hp

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The HiTrap SP HP is a pre-packed ion exchange chromatography column designed for fast and efficient protein purification. It features a strong cation exchange resin with a high dynamic binding capacity, enabling the capture and separation of positively charged proteins. The column is suitable for a variety of purification applications and can be used with various chromatography systems.

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HiTrap SP HP column (52 citations) HiTrap SP (25 citations) HiTrap SP HP cation exchange column (14 citations) HiTrap SP HP 5 mL column (6 citations) HiTrap™ SP HP 5 ml (4 citations) HiTrap SP/Q HP columns (3 citations) HiTrap SP HP cation (3 citations)

65 protocols using hitrap sp hp

1

Recombinant Baculovirus Protein Purification

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Recombinant baculoviruses encoding POLγB and the different POLγA versions were expressed in Sf9 cells32 (link). These recombinant proteins all lacked the N-terminal mitochondrial targeting sequence and carried a carboxy-terminal 6 × His-tag. The proteins were purified over HIS-Select Nickel Affinity Gel (Sigma-Aldrich) and HiTrap Heparin HP (GE Healthcare), followed by HiTrap SP HP or HiTrap Q HP columns (GE Healthcare), depending on the net electrical charge of the protein. MtSSB lacking the N-terminal mitochondrial targeting sequence was expressed in insect cells and purified over DEAE Sepharose Fast Flow (GE Healthcare), HiTrap Heparin HP and HiTrap SP HP, followed by gel filtration using HiLoad Superdex 200 (GE Healthcare).
Persson Ö., Muthukumar Y., Basu S., Jenninger L., Uhler J.P., Berglund A.K., McFarland R., Taylor R.W., Gustafsson C.M., Larsson E, & Falkenberg M. (2019). Copy-choice recombination during mitochondrial L-strand synthesis causes DNA deletions. Nature Communications, 10, 759.
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2

Purification of Mitochondrial Replication Proteins

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Recombinant baculoviruses encoding TWINKLE, mitochondrial Lig3, POLγB and the different POLγA versions were expressed in Sf9 cells21 (link). These recombinant proteins all lacked the N-terminal mitochondrial targeting sequence and carried a carboxy-terminal 6 × His-tag. The proteins were purified over HIS-Select Nickel Affinity Gel (Sigma-Aldrich) and HiTrap Heparin HP (GE Healthcare), followed by HiTrap SP HP or HiTrap Q HP columns (GE Healthcare), depending on the net electrical charge of the protein. MtSSB lacking the N-terminal mitochondrial targeting sequence was expressed in insect cells and purified over DEAE Sepharose Fast Flow (GE Healthcare), HiTrap Heparin HP and HiTrap SP HP, followed by gel filtration using HiLoad Superdex 200 (GE Healthcare).
Macao B., Uhler J.P., Siibak T., Zhu X., Shi Y., Sheng W., Olsson M., Stewart J.B., Gustafsson C.M, & Falkenberg M. (2015). The exonuclease activity of DNA polymerase γ is required for ligation during mitochondrial DNA replication. Nature Communications, 6, 7303.
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3

Recombinant Yeast Dot1 Protein Purification

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Plasmid DNA of pET15b-His-3C-Dot1 (158-582) was generated using Gibson Assembly (NEB). Yeast Dot1 plasmid DNA was transformed into ONESHOT™BL21(DE3) (ThermoFisher) competent cells and grown in 2xYT-Amp media. Yeast Dot1 was expressed as soluble protein by inducing with 0.1mM IPTG for 16 hours at 18°C upon the culture reaching OD600 = 0.4-0.6. Cells were harvested (Sorvall LYNX6000) and lysed (AvestinEmulsiflexC3), and the protein was purified through Ni-NTA agarose beads (Qiagen) (Lysis Buffer: 500 mM NaCl, 20 mM HEPES pH 7.5, 20 mM Imidazole, 1 mM BME, 1× Protease Inhibitor / Elution Buffer: 500 mM NaCl, 20 mM HEPES pH 7.5, 300 mM Imidazole, 1 mM BME). Eluted yeast Dot1 protein was digested with 3C PreScission Protease (GE Healthcare) while dialyzed in digestion buffer (Digestion Buffer: 100 mM NaCl, 20 mM HEPES pH 7.5, 1 mM DTT) overnight at 4°. After which, sample was purified through HiTrap SP HP (GE Healthcare) liquid chromatography column (Buffer A: 100 mM NaCl, 20 mM HEPES pH 7.5, 1 mM DTT / Buffer B: 1 M NaCl, 20 mM HEPES pH 7.5, 1 mM DTT). Selected fractions were further purified through HiLoad Superdex 200 (GE Healthcare) size-exclusion liquid chromatography column (S200 Buffer: 150 mM NaCl, 10 mM Tris pH 7.5, 5 mM DTT). Purified yeast Dot1 protein was then concentrated, flash frozen in liquid nitrogen and stored in −80°C for future use.
Valencia-Sánchez M.I., De Ioannes P., Wang M., Vasilyev N., Chen R., Nudler E., Armache J.P, & Armache K.J. (2019). Structural basis of Dot1L stimulation by histone H2B lysine 120 ubiquitination. Molecular cell, 74(5), 1010-1019.e6.
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4

Purification and Production of Cas9 Protein

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Streptococcus pyogenes Cas9 (pMJ915, Addgene #69090) with two nuclear localization signal peptides and an HA tag at the C terminus was expressed in Rosetta2 DE3 (UC Berkeley Macrolab) cells. Cell pellets were sonicated, clarified, Ni2+-affinity purified (HisTraps, GE life sciences), TEV cleaved, cation-exhanged (HiTrap SP HP, GE life sciences), size excluded (Sephacryl S-200, GE life sciences) and eluted at 40 μM in 20 mM HEPES KOH pH 7.5, 5% glycerol, 150 mM KCl, 1 mM dithiothreitol (DTT)20 (link). sgRNAs were generated by HiScribe (NEB E2050S) T7 in vitro transcription using PCR-generated DNA as a template (dx.doi.org/10.17504/protocols.io.dm749m). Complete sequences for all sgRNA templates can be found in Supplementary Data 2.
Richardson C.D., Ray G.J., Bray N.L, & Corn J.E. (2016). Non-homologous DNA increases gene disruption efficiency by altering DNA repair outcomes. Nature Communications, 7, 12463.
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5

Purification of Bacteriophage T4 Lysozyme

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The pellet of cells (3 g) that expressed the pET-gE plasmid was resuspended in 25 mL of buffer E (0.1 M sodium phosphate buffer, pH 6.6, 0.2 M NaCl, 10 mM MgCl2 and 1 mM CaCl2). Then, 0.5 mL of chloroform was added to this mixture and the suspension was stirred for 30 min. To reduce the viscosity, 1 mL of 1 M MgCl2 and a few grains of DNase I were added, and the mixture was stirred for an additional 1.5 h. The lysates were centrifuged for 30 min at 28,000× g (the R18A rotor) to remove cell debris. The supernatants were dialyzed into buffer F (50 mM Tris pH7.25, 1 mM EDTA). The dialyzed sample was applied to the HiTrap CM FF (5 mL, GE healthcare) cation exchange column, pre-equilibrated with buffer F. The bound material was eluted by 50–300 mM NaCl linear gradient with the same buffer. Fractions containing T4L were pooled, dialyzed against 50 mM sodium phosphate buffer (pH 5.8), and then run through a HiTrap SP HP (1 mL, GE healthcare) cation exchange column equilibrated with the same buffer. T4L was eluted with buffer G (0.1 M sodium phosphate, pH 6.6, 0.55 M NaCl).
Kanamaru S., Uchida K., Nemoto M., Fraser A., Arisaka F, & Leiman P.G. (2020). Structure and Function of the T4 Spackle Protein Gp61.3. Viruses, 12(10), 1070.
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6

Expression and Purification of Isotopically-Labeled PTP1B

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The PTP1B construct of amino acids 1-301 was transformed into BL21(DE3) cells and expressed as previously described for 20 hours at 25 °C.31 (link) PTP1B cells were grown in perdeuterated M9 minimal medium supplemented with 15NH4Cl (1.0 g/L). Additionally, 60 mg/ml α-ketobutyric acid and 100mg/ml α-ketoisovaleric acid (Cambridge Isotope Labs) was added half an hour before induction time for 13C labeling of the methyl groups of isoleucine, leucine and valine residues. Cells were allowed to reach an OD600 of 0.8 – 1.0 before induction with 0.5 mM IPTG. PTP1B cell pellets were resuspended in a buffer containing 20 mM Bis Tris, 20 mM imidazole, 3 mM dithiothreitol (DTT), 10% w/v glycerol, 1 mM ethylenediaminetetraacetic acid (EDTA) at pH 6.5. Cells were lysed by ultrasonication, centrifuged, and the supernatant was applied to HiTrap Q HP and HiTrap SP HP columns (GE Healthcare) using a buffer of 20 mM Bis Tris, 20 mM imidazole, 3 mM DTT, 10% w/v glycerol, 1 mM EDTA, 0.5 M NaCl at pH 6.5 and eluted with a NaCl gradient. The protein sample was concentrated to 0.18 – 0.24 mM for NMR studies in a 50 mM HEPES buffer containing 150 mM NaCl, 0.5 mM TCEP, 7% D2O, and 0.03% NaN3 at pH 6.8.
Cui D.S., Lipchock J.M., Brookner D, & Loria J.P. (2019). Uncovering the molecular interactions in the catalytic loop that modulate the conformational dynamics in protein tyrosine phosphatase 1B. Journal of the American Chemical Society, 141(32), 12634-12647.
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7

Synthesis of K48 and K63 Polyubiquitin Conjugates

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K48-linked polyUb conjugates were obtained from reaction with UBE2R1. UbK48C (0.8 mM) and 1 mM of Ub(6×His) were reacted with 20 μM UBE2R1, 3 mM TCEP, 20 mM ATP, 20 mM MgCl2 and 1 μM UBA1 in 50 mM Tris–HCl, pH 8.0 at 30 °C for 18 hours. K48CUb−48Ub(6×His) was purified using a 5-mL His-Trap followed by a HiLoad 26/600 Superdex 75 column in 25 mM Tris–HCl, 300 mM NaCl and 3 mM TCEP, pH 7.5. Unanchored wild-type K48-linked conjugates were formed under the same reaction conditions using 1 mM UbWT and 0.8 mM His-TEV-UbΔGG, followed by Ni-NTA pulldown, TEV cleavage and an additional Ni-NTA step to remove TEV protease. The final cation exchange step used a 0–35% gradient buffer A (50 mM ammonium acetate, pH 4.5) and buffer B (50 mM ammonium acetate, 500 mM NaCl, pH 4.5) over 60 CV on a 5-mL HiTrap SP HP (GE Life Sciences) to separate Ub1 to Ub4. Unanchored wild-type K63-linked polyUb was obtained from identical conditions substituting UBE2R1 for 20 μM UBE2N/UBE2V2.
Nakasone M.A., Majorek K.A., Gabrielsen M., Sibbet G.J., Smith B.O, & Huang D.T. (2022). Structure of UBE2K–Ub/E3/polyUb reveals mechanisms of K48-linked Ub chain extension. Nature Chemical Biology, 18(4), 422-431.
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8

PARP14 and PARP9 Macrodomain Purification

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BL21(DE3)-R3-pRARE cells were transformed with PARP14 and PARP9 macrodomain encoding constructs and grown at 37°C in LB medium supplemented with appropriate antibiotics until optical density at 600 nm (OD600) 0.5 to 0.6, then cooled to 18°C, and supplemented with 0.5 mM isopropyl-β-d-thiogalactopyranoside at an OD600 of 0.8 to induce protein expression overnight. Cells were harvested by centrifugation, resuspended in lysis buffer [50 mM Hepes (pH 7.5), 500 mM NaCl, 20 mM imidazole, 5% glycerol, 0.5 mM tris(2-carboxyethyl)phosphine (TCEP), and 1:2000 Calbiochem protease inhibitor cocktail set III), and lysed by sonication. Proteins were purified by Ni2+–nitrilotriacetic acid chromatography (Jena Bioscience) and eluted stepwise in binding buffer with 40 to 250 mM imidazole. Proteins were further purified by size exclusion chromatography (Superdex 75, GE HealthCare) in a buffer consisting of 50 mM Hepes (pH 7.5), 300 mM NaCl, 5% glycerol, and 0.5 mM TCEP. PARP14 MD1 was additionally purified by ion exchange chromatography using a HiTrap SP HP (5 ml; GE HealthCare) equilibrated in 25 mM Hepes (pH 7.5), 75 mM NaCl, and 0.5 mM TCEP. The purity of protein preparations was assessed using SDS-PAGE and Coomassie Brilliant Blue staining, and aliquots were stored at −80°C until use.
Đukić N., Strømland Ø., Elsborg J.D., Munnur D., Zhu K., Schuller M., Chatrin C., Kar P., Duma L., Suyari O., Rack J.G., Baretić D., Crudgington D.R., Groslambert J., Fowler G., Wijngaarden S., Prokhorova E., Rehwinkel J., Schüler H., Filippov D.V., Sanyal S., Ahel D., Nielsen M.L., Smith R, & Ahel I. (2023). PARP14 is a PARP with both ADP-ribosyl transferase and hydrolase activities. Science Advances, 9(37), eadi2687.
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9

FH Binding to FHbp by ELISA

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We assayed binding of serum FH to FHbp by ELISA, which was performed as previously described [5 (link)]. Briefly, the wells of a microtiter plate were coated with recombinant FHbp ID 1 (2 μg/ml in PBS; 100 μl per well), which was purified as described previously [14 (link)] with the addition of a second ion exchange chromatography step (HiTrap SP HP; GE Life Sciences). The plate was incubated at 4°C overnight. After washing and blocking, two-fold serial dilutions of macaque sera were added to the wells starting at a dilution of 1:100. After incubation for 2 h at room temperature, bound FH was detected with a sheep anti-human FH antibody (1:7,000; Abcam). The bound sheep IgG was detected with donkey anti-sheep IgG conjugated to alkaline phosphatase (AP) (Sigma-Aldrich; 1:5,000) for 1 h at room temperature. The assays were performed in three independent experiments, each in duplicate and every plate had the same human serum sample as a positive control.
Konar M., Beernink P.T, & Granoff D.M. (2015). A Newly-Identified Polymorphism in Rhesus Macaque Complement Factor H Modulates Binding Affinity for Meningococcal FHbp. PLoS ONE, 10(8), e0135996.
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10

Purification and Characterization of TtAgo

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Wild-type or mutant TtAgo (TtAgoD478A,D546A or TtAgoDM) cloned and expressed from pET-SUMO (Invitrogen K30001) in E. coli BL21-DE3 was purified as described (Wang et al., 2008a (link)) except: (1) After cleavage and removal of the 6×His-SUMO tag, wild-type or mutant TtAgo was additionally purified by HiTrap SP HP (GE Healthcare) chromatography, dialyzed against 3 × 2 L 20 mM HEPES-KOH pH 7.4, 250 mM potassium acetate, 3 mM magnesium acetate, 0.1 mM 2,2′,2′′,2′′′-(ethane-1,2-diyldinitrilo)tetraacetic acid, 10% glycerol (w/v), 5 mM DTT (TtAgo) or 30 mM HEPES-KOH, pH 7.4, 250 mM potassium acetate, 1 mM DTT, 0.01% Igepal CA-630, 20% [v/v] glycerol (TtAgoDM). Purified protein was aliquoted into tubes, flash frozen in liquid nitrogen, and stored at −80°C. Protein was quantified by Bradford Assay using BSA as standard or by amino acid analysis (Protein Structure Core Facility, University of Nebraska Medical Center). Purification of TtAgo with magnesium buffers yields guide-free TtAgo and purification with manganese containing buffers retains endogenous guides (Swarts et al., 2014 (link)). TtAgo, purified as above with magnesium containing buffers, has an A260/A280 ratio of 0.56, indicating it to be free of nucleic acids. SYBR Gold (Invitrogen) staining did not detect nucleic acids.
Jolly S.M., Gainetdinov I., Jouravleva K., Zhang H., Strittmatter L., Bailey S.M., Hendricks G.M., Dhabaria A., Ueberheide B, & Zamore P.D. (2020). Thermus thermophilus Argonaute Functions in the Completion of DNA Replication. Cell, 182(6), 1545-1559.e18.
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