C2 RNA cleavage was performed as described previously26 (link),27 (link) with minor changes. Full-length ScLas1-Grc3 variants (0–0.8 μM) were incubated with 1 mM EDTA and 50 nM fluorescein labeled C2 RNA mimic (5′-GUCGUUUUAGGUUUUACCAACUGCGGC/36-FAM/−3′) in the absence and presence of 2 mM nucleotide (ATP). Contaminating nucleotides were removed from commercial ATP (Sigma; A2383) using previously established nucleotide purification protocols60 ,61 . Briefly, ATP (6 mg) was resuspended in 50 mM (NH4)HCO3 and bound to a MonoQ 4.6/100 PE (GE Healthcare) column equilibrated with 50 mM (NH4)HCO3. ATP was eluted using a 50–500 mM (NH4)HCO3 linear gradient over 45 mL. ATP fractions were lyophilized and resuspended to 10 mM using water. Unless specified otherwise, reactions were incubated for 1 hour at 37°C and quenched with urea-loading dye. Samples were resolved on 15% polyacrylamide (8 M urea) gels in 1x tris-borate-EDTA buffer and visualized using a Typhoon FLA 9500. Representative gels are shown from three independent replicates.
Monoq 4.6 100 pe
Manufactured by GE Healthcare
The MonoQ 4.6/100 PE is a lab equipment product from GE Healthcare. It is a strong anion exchange chromatography column designed for the purification of proteins, peptides, and other biomolecules. The column has a bed volume of 1.7 mL and a maximum flow rate of 5 mL/min.
Automatically generated - may contain errors
Lab products found in correlation
Bio rex 70, by Bio-Rad (1 mentions)
Cy5 maleimide monoreactive dye, by GE Healthcare (1 mentions)
Hispur ni nta, by Thermo Fisher Scientific (1 mentions)
Superose 6 10 300 gl column, by GE Healthcare (1 mentions)
Akta purifier fplc system, by GE Healthcare (1 mentions)
Ni sepharose fast flow resin, by GE Healthcare (1 mentions)
Superose 12 hr10 30, by GE Healthcare (1 mentions)
Phenyl superose hr5 5, by GE Healthcare (1 mentions)
Ni nta resin, by Thermo Fisher Scientific (1 mentions)
Superdex 200 10 300 gl, by GE Healthcare (1 mentions)
8 protocols using monoq 4.6 100 pe
1
Fluorescent RNA Cleavage Assay
2
Purification and Labeling of Histones
3
Purification of Wdr78 Protein Complex
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Eight-week-old mouse testis were homogenized in HMDEK buffer (30 mM HEPES, pH 7.4, 5 mM MgSO4, 1 mM DTT, 0.5 mM EGTA, 1% glycerol, 25 mM KCl) containing 0.1% NP-40, 10 μg/ml nocodazole, and proteinase inhibitors (PMSF and cocktail) (Sloboda, 2009 (link)). The lysates were cleared by centrifugation at 13000 rpm at 4°C for 30 min and filtered through a 0.22-μm filter. The solution was loaded on a prepacked Superose 6 10/300 GL column (GE Healthcare) on a GE AKTA Purifier FPLC system at 4°C. Elution was performed with HMDEK buffer at a flow rate of 0.5 ml/min. Fractions #12–#14 that were abundant in Wdr78 were pooled and applied to an anion-exchange column (Mono Q 4.6/100 PE, GE Healthcare). The bound proteins were eluted with a linear salt gradient of 0.2–1.0 M KCl in HMDEK buffer at a flow rate of 0.5 ml/min. The 0.5-ml fractions were collected for immunoblotting.
4
Expression and Purification of Recombinant Human PAD4
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Full length human PAD4 with N-terminal His-tag was expressed in E. coli BL21 (DE3) cells using the pET-16b vector. HisTag-PAD4 expression was induced with 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) at OD600nm = 0.6 and the culture was incubated at 25 °C for 16 h. After this time cells were harvested, lysed and soluble protein fraction was applied on Ni Sepharose Fast Flow resin (GE Healthcare, Life Science, USA). Fractions containing HisTag-PAD4 were further purified using combination of ion exchange and gel filtration chromatography on MonoQ 4.6/100PE and HiLoad Superdex S75 16/600 columns respectively (GE Healthcare, Life Science). Finally, HisTag-PAD4 was concentrated and frozen in storage buffer (50 mM Hepes pH 7.5 with 0.25 M NaCl, 3% glycerol). PAD4 activity was determined on N-α-benzoyl-L-arginine ethyl ester hydrochloride (BAEE) substrate with colorimetric assay [33 (link)].
5
Purification of MglB Mutants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
For MglB mutants, the protocol was followed the same as MglB purification. However, following Superdex 75, the protein was impure. Hence, it was injected in MonoQ 4.6/100 PE (GE HealthCare). Buffers used for binding and elution were Buffer A (50 mM Tris [pH 8.0], 50 mM NaCl) and Buffer B (50 mM Tris [pH 8.0], 1 M NaCl), respectively. A linear gradient of Buffer A ranging from 0% to 50% Buffer B over 20 column volumes was injected, and the fractions containing the protein were pooled and concentrated.
6
Xenopus laevis Oocyte Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Xenopus laevis adult females (Centre de Ressources Biologiques Xenopes, CNRS, France) were bred and maintained according to current French guidelines in the conventional IBPS aquatic animal facility, with authorization: Animal Facility Agreement: #A75-05-25. All experiments were subject to ethical review and approved by the French Ministry of Higher Education and Research (reference APAFIS#14127-2018031614373133v2).
All reagents, unless otherwise specified, were from Sigma. Okadaic acid (OA), magnetic GSH-beads and Co-beads were purchased from Enzo Life Sciences, Promega and Clontech Laboratories respectively. Uno Q-25 was purchased from Bio-Rad, Mono Q 4.6/100PE, Phenyl-Superose HR5/5 and Superose 12 HR10/30 were from GE Healthcare.
All reagents, unless otherwise specified, were from Sigma. Okadaic acid (OA), magnetic GSH-beads and Co-beads were purchased from Enzo Life Sciences, Promega and Clontech Laboratories respectively. Uno Q-25 was purchased from Bio-Rad, Mono Q 4.6/100PE, Phenyl-Superose HR5/5 and Superose 12 HR10/30 were from GE Healthcare.
7
Fluorescent RNA Cleavage Assay
8
Recombinant H6 Hemagglutinin Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The genes encoding the ectodomains of A/Taiwan/2/2013(H6N1) (TW H6) and A/chicken/Guangdong/S1311/2010(H6N6) (GD H6) HA proteins were assembled [57 (link)] from oligos ordered from Integrated DNA Technologies. The H6 HA genes were inserted into pFastBac1 vector that was modified to contain a C-terminal T4 fibritin (foldon) and His6-tag [58 (link),59 (link)]. The recombinant baculoviruses were made according to the manufacturer’s instruction (Invitrogen). High Five cells were infected at a multiplicity of infection of ~1.0 for 40 hours and the culture was harvested. The supernatant was dialyzed against 20 mM Tris, pH 7.5, 50 mM NaCl followed by incubating with Ni-NTA resin (Thermo Scientific). The HA-bound resin was washed with wash buffer (20 mM Tris, pH 7.5, 50 mM NaCl and 15 mM imidazole) and digested by trypsin (at a weight ratio of about 1:1000) at room temperature overnight to remove the C-terminal foldon and His6-tag, which also cleaved HA into HA1 and HA2. The cleaved HA was further purified by Mono Q 4.6/100 PE and Superdex 200 10/300 GL (GE Healthcare).
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!