For reconstitution and purification of U1 snRNP, full-length U1 snRNA was transcribed in large scale in vitro using run off transcription from T7 promoter and treated with DNase I. U1 snRNP was assembled as described before (30 ) and purified by anion exchange chromatography (Mono Q; Cytiva) using a KCl gradient (from 250 mM KCl through 1M KCl). Particles were flash-frozen in liquid N2 and stored at –80°C in single-use aliquots.
Monoq
Manufactured by Cytiva
Sourced in United States
The MonoQ is a high-performance anion exchange chromatography column designed for the purification of biomolecules. It features a porous, polymeric matrix that provides high surface area and efficient separation. The column can be used to purify a variety of charged biomolecules, including proteins, peptides, and nucleic acids.
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Lab products found in correlation
5 protocols using monoq
1
Purification of U1 snRNP Complex
2
Overexpression and Purification of NC Proteins
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The 55-amino acid NCNL4.3 protein (MQKGN FRNQR KTVKC FNCGK EGHIA KNCRA PRKKG CWKCG KEGHQ MKDCT ERQAN, 6369 Da) was overexpressed and purified as previously described (37 (link)). The codon-optimized gene sequence encoding the 54-amino acid NCMAL protein (MQRGN FKGQK RIKCF NCGKE GHLAR NCRAP RKKGC WKCGK EGHQM KDCTE RQAN) was ordered from Genewiz (Morrisville, NC) and inserted into the pLATE11 plasmid, using the aLICator LIC cloning system (Thermo Fisher Scientific). The pLATE11 plasmid was transformed into BL21(DE3) expression cells. The protein was purified under nondenaturing conditions. Briefly, cells were lysed by microfluidization, followed by purification with ion exchange chromatography using MonoQ and MonoS columns (Cytiva). After elution from the SP column, concentrated fractions were furthered purified and buffer exchanged with size-exclusion chromatography on the Superdex 30 prep grade column (Cytiva). The molecular weight of NCMAL was verified by mass spectrometry to be 6521.
3
Purification of recombinant PTP1B protein
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BL21(DE3) cells transformed with pGEX-4T encoding GST-(TEV)-PTP1B (2-321) were grown in S-broth plus 100 ug/ml Ampicillin at 37 °C, 180 rpm. At OD600 of 1, expression was induced with 1 mM IPTG at 18 °C, and growth continued overnight. Cells were harvested by centrifugation and resuspended in 20 mM Tris pH7.5, 300 mM NaCl, 2 mM TCEP, 1 mM PMSF, 5 mM Phenyl phosphate, DNase I and ~20 mg Lysosome. Cells were lysed by sonication, clarified by centrifugation, and the supernatant was applied to Glutathione Agarose (UBPBio). Beads were washed with 20 column volumes of the resuspension buffer, and PTP1B was eluted by TEV digest at 4°C overnight in 20 mM Tris, 300 mM NaCl, 2 mM TCEP. The eluate was concentrated and applied to Superdex200 size exclusion column (Cytiva) equilibrated in 20 mM Tris pH7.5, 300 mM NaCl, 2 mM TCEP. The Purest fractions as analyzed by SDS-PAGE were pooled and desalted into 20 mM Tris-HCl, pH 8.5, 50 mM NaCl before being applied to a MonoQ (HR5/5 Cytivia) equilibrated in 20 mM Tris-HCl, pH 8.5, 50 mM NaCl. PTP1B was eluted with a gradient from 20 mM Tris-HCl pH 8.5, 50 mM NaCl to 20 mM Tris-HCl pH 8.5, 700 mM NaCl over 20 column volumes. The cleanest fractions were pooled and concentrated to A280 12.6, TCEP was added to 2 mM and the protein was snap frozen in liquid nitrogen and stored at −80 °C.
4
Recombinant Expression and Purification of Truncated Human ACE2
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DNA encoding the truncated human ACE2 ectodomain (residues 19–613) with a C-terminal AVI-tag and 6xHis-tag was synthesized (IDT) and subcloned into a pHLSec expression plasmid. Plasmid DNA was used for transient expression in Expi293F cells using ExpiFectamine 293 Transfection Kits (Thermo Fisher Scientific). Expression supernatant was harvested at day 6 and dialyzed into 10 mM Tris pH 8.0. ACE2 was subsequently purified using weak anion exchange (DEAE Sepharose, Cytiva), followed by size-exclusion chromatography (Superdex 200, Cytiva). Protein was then biotinylated enzymatically using BirA enzyme and further purified using strong anion exchange (MonoQ, Cytiva).
5
Recombinant CbrR Protein Purification
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To express a His-tagged recombinant protein, cbrR was amplified from pCAF101 using primers CF112 and CF113 and ligated into pET-20b(+) to add a C-terminal His6-tag for purification purposes, creating pCAF107. Protein was affinity-purified by binding to Ni-NTA agarose (Thermo Scientific, Waltham, MA, USA) and subsequently further purified via ion-exchange chromatography on a Mono Q (Cytiva, Marlborough, MA, USA) column (binding buffer: 20 mM Tris-HCL, pH7.5; 1 mM DTT; elution buffer: 20 mM Tris-HCL, pH7.5; 1 mM DTT; 1 M NaCl) and dialyzed into PBS for polyclonal rabbit antiserum generation (Cocalico Biologicals, Stevens, PA, USA) (Supplementary Figure S1 ). Western blots were performed on 1 μg total protein from whole-cell lysates of all three strains using antisera against CbrR, FlaA (each at 1:4000 dilution) or FliF (at 1:2000 dilution) as primary antibodies, and goat anti-rabbit HRP-conjugate IgG (1:20,000) or goat anti-mouse HRP-conjugate IgG (1:100,000) as secondary antibodies. SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Thermo Scientific, Waltham, MA, USA) was used to develop Western blot images. RNA-Seq experiments (manuscript in preparation) show that FliF is expressed equally in these strains; therefore, anti-FliF antibodies were used to demonstrate equivalent loading of the protein samples.
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