For analysis of endogenous protein profiles in HAP1 cells, nuclear extracts (high salt) were first dialyzed against BP100 (50 mM potassium phosphate pH 6.8, 100 mM NaCl, 1 mM EDTA, 1 mM DTT, protease inhibitors) and clarified by high-speed centrifugation. Samples were then purified by ion exchange chromatography using a HiTrap SP HP 5 ml column (GE Healthcare). Elution was performed by step elution with increasing NaCl concentration. In all, 500 mM elution was concentrated 5× time on centricon (Millipore, cut-off 10 kDa) and analyzed on Superose 6 PC3.2 increase column (GE Healthcare). The native molecular size markers used for column calibration were thyroglobulin (669 kDa), ferritin (440 kDa), and aldolase (158 kDa).
Hitrap sp hp 5 ml column
Manufactured by GE Healthcare
The HiTrap SP HP 5 mL column is a pre-packed, ready-to-use ion exchange chromatography column designed for purification of proteins and other biomolecules. The column is packed with a sulfopropyl (SP) cation exchange medium and has a bed volume of 5 mL. It is intended for use in protein purification workflows.
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Lab products found in correlation
Hiload 16 60 superdex 75, by GE Healthcare (2 mentions)
Histrap ff 5 ml column, by GE Healthcare (2 mentions)
Centricon, by Merck Group (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Bis tris sds page gel, by Thermo Fisher Scientific (1 mentions)
Protein l column, by GE Healthcare (1 mentions)
Hiload 16 60 superdex 75 column, by GE Healthcare (1 mentions)
6 protocols using hitrap sp hp 5 ml column
1
Endogenous Protein Profiling in HAP1 Cells
2
Recombinant Histone Purification Protocol
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Histones were expressed and purified as described in Kilic et al. (2015) (link). Briefly, individual wild-type human histones were cloned into pet15b plasmid vectors and expressed in BL21 DE3 plysS cells. Cells were grown in LB media containing 100 μg/mL ampicillin and 35 μg/mL chloramphenicol at 37°C until the OD600 reached 0.6. Expression was induced by IPTG addition to a final concentration of 0.5 mM. After 3 h expression, cells were harvested by centrifugation and resuspended in lysis buffer (20 mM Tris pH 7.5, 1 mM EDTA, 200 mM NaCl, 1 mM βMe, Roche protease inhibitor) and frozen. Cells were lysed by freeze-thawing and sonication. Inclusion bodies were harvested by centrifugation. The inclusion body pellet was washed once with 7.5 mL of lysis buffer containing 1% Triton and once without. Inclusion body pellets were resolubilized in resolubilization buffer (6 M GdmCl, 20 mM Tris pH 7.5, 1 mM EDTA, 1 mM βMe) and dialyzed into urea buffer (7 M urea, 10 mM Tris, 1 mM EDTA, 0.1 M NaCl, 5 mM 1 mM βMe, pH 7.5). Histones were purified by cation exchange chromatography using a HiTrap SP HP 5 mL column (GE Healthcare). Fractions were analyzed by SDS-PAGE and pooled, followed by dialysis into water and lyophilization. Final purification was performed by preparative RP-HPLC. Purified histones were lyophilized and stored at −20°C until used for octamer refolding.
3
Expression and Purification of TtCuA Proteins
4
Purification of Human eIF1 and eIF1A
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Plasmids encoding human eIF1 and eIF1A with an N-terminal His6-MBP tag separated by a TEV cleavage site were transformed, expressed, and purified as mentioned36 (link). Briefly, the proteins were expressed in E. coli BL21-CodonPlus (DE3)-RIPL in 2xYT medium at 18°C. After 16 h, cells were harvested by centrifugation, resuspended in lysis buffer (50 mM HEPES-KOH pH 7.6, 800 mM KCl, 5 mM MgCl2, 40 mM imidazole, 10% (w/v) glycerol, 0.5 mM TCEP and protease inhibitors) and lysed using sonification (Sonifer SFX 550). The cleared lysate was loaded onto a HisTrap FF 5 ml column (GE Healthcare) and eluted proteins were incubated with TEV protease, dialysed (3.5 kDa MWCO) at 4°C overnight and run on the HiTrap SP HP 5 ml column to remove cleaved MBP-fusion protein. In case of eIF1, the sample was further purified via size-exclusion chromatography on a HiLoad 16/60 Superdex75 (GE Healthcare), buffer exchanging the sample to the storage buffer (40 mM HEPES-KOH pH 7.6, 200 mM KCl, 5 mM MgCl2, 10% (w/v) glycerol, 1 mM TCEP). The final protein was flash-frozen in liquid nitrogen and stored until further use at −80°C.
5
Purification of Recombinant Antibodies and Lactoferrin
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Full-length DC25 (IgG1) and MN423 (IgG2b) antibodies were purified on a 5 mL HiTrap™ Protein A column, DC25 and DC11 Fabs (IgG1) were purified on a 5 mL HiTrap™ protein G column and MN423 Fab (IgG2b) was purified on a 1 mL Protein L column (all chromatography media were from GE Healthcare, Chicago, IL, USA). The final polishing of recombinant Fabs was performed on a HiLoad Superdex 75 16/60 column (GE Healthcare) in 10 mM Tris pH 7.2 and 50 mM NaCl. Fractions were checked on 12% SDS PAGE, pooled and concentrated through ultrafiltration. Purification of recombinant lactoferrin (rhLf) was performed using cation exchange chromatography. The medium of rhLf expressing CHO cells was adjusted to pH 7 by 100 mM NaOH and pre-cleared through centrifugation at 21,000× g for 10 min at 4 °C. The supernatant was filtered through a 0.22 mm Acrodisc Syringe Filter and loaded on a HiTrap SP HP 5 mL column (GE Healthcare) primed using a mobile phase of 20 mM sodium phosphate pH 7. Protein elution was accomplished with a gradient of 1.5 M NaCl in the mobile phase buffer, and rhLf was eluted as a symmetric peak fraction at about 0.7 M NaCl. The concentration of pure proteins was determined from the absorbance at 280 nm.
6
Expression and Purification of eIF1 and eIF1A
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Plasmids encoding human eIF1 and eIF1A with an N-terminal His 6 -MBP tag separated by a TEV cleavage site were transformed, expressed, and purified as mentioned 36 . Briefly, the proteins were expressed in E. coli BL21-CodonPlus (DE3)-RIPL in 2xYT medium at 18°C. After 16 h, cells were harvested by centrifugation, resuspended in lysis buffer (50 mM HEPES-KOH pH 7.6, 800 mM KCl, 5 mM MgCl 2 , 40 mM imidazole, 10% (w/v) glycerol, 0.5 mM TCEP and protease inhibitors) and lysed using sonification (Sonifer SFX 550). The cleared lysate was loaded onto a HisTrap FF 5 ml column (GE Healthcare) and eluted proteins were incubated with TEV protease, dialysed (3.5 kDa MWCO) at 4°C overnight and run on the HiTrap SP HP 5 ml column to remove cleaved MBP-fusion protein. In case of eIF1, the sample was further purified via size-exclusion chromatography on a HiLoad 16/60 Superdex75 (GE Healthcare), buffer exchanging the sample to the storage buffer (40 mM HEPES-KOH pH 7.6, 200 mM KCl, 5 mM MgCl 2 , 10% (w/v) glycerol, 1 mM TCEP). The final protein was flash-frozen in liquid nitrogen and stored until further use at -80°C.
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