Prior to the addition of TEV protease, a buffer exchange was carried out using a 10 kDa MWCO centrifugal filter (Millipore, Cat. # UFC8010) to remove the imidazole present in the elution buffer. The cleavage reaction using TEV protease (Genscript, Cat. # Z03030) was subsequently carried out at 4°C for 16 hours. Equilibrated Ni-NTA resin was added to the products to bind the MBP-His tag and the His-tagged TEV protease (Genscript, Cat. # Z03030) while allowing the purified FAM210A-dMTS cleaved product to be collected in the flowthrough. TEV was added based on the quantity of the protein concentration that needed to be cleaved. We used 1 unit of the TEV protease per 3 μg of the fusion protein.
Tev protease
Manufactured by GenScript
Sourced in United States
TEV protease is a lab equipment product that serves as a highly specific and efficient enzyme for cleaving recombinant fusion proteins. It recognizes and cleaves the amino acid sequence ENLYFQ↓G, allowing for the separation of the target protein from affinity tags or other fusion partners.
Automatically generated - may contain errors
Lab products found in correlation
Tev protease, by Merck Group (1 mentions)
Tetramethyl rhodamine 5 dutp, by Roche (1 mentions)
Halocarbon oil 700, by Merck Group (1 mentions)
Bf100 58 10, by Sutter Instruments (1 mentions)
Hiload 26 60 superdex 200 pg, by GE Healthcare (1 mentions)
Ni2 nta agarose, by Qiagen (1 mentions)
Deltavision software, by GE Healthcare (1 mentions)
Mouse monoclonal anti gfp antibody, by Roche (1 mentions)
Deltavision microscope, by GE Healthcare (1 mentions)
7 protocols using tev protease
1
Affinity-based Protein Purification
2
Recombinant MeABL5 Protein Production
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The open reading frames (ORFs) of MeABL5 were amplified with added Hind III and Xho I restriction sites, and then fused to the maltose-binding protein (MBP) in the pET28a-MBP vector (GenScript, Nanjing, China), which generated the pET28a-MBP-MeABL5. The pET28a-MBP vector contains an MBP that can promote the folding of target proteins and increase their expression level and solubility in prokaryotic expression systems. The MBP-tagged MeABL5 protein was expressed in Escherichia coli BL21 (DE3) by inducing with 1.0 mM isopropyl-β-D-thiogalactopyranoside (IPTG) at 37°C for 4 h, followed by purification using a Ni column (GenScript, Nanjing, China) according to the manufacturers’ instructions. The MBP-tagged protein was then cleaved with TEV Protease (GenScript, Nanjing, China) to remove the MBP affinity tag from the fusion protein. The recombinant MeABL5 protein was dialyzed and sterilized by a 0.22-μm filter before being stored in aliquots. The concentration was determined by Bradford protein assay with bovine serum albumin (BSA) as a standard. The protein purity and molecular weight were determined by standard SDS-PAGE along with Western blot.
3
Germline Microinjection in C. elegans
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Germline injections were performed in young adult worms at 18–24 h post L4 immobilised in 2% agarose pads and covered with Halocarbon oil 700 (Sigma) using a Narishige IM-31 pneumatic microinjector attached to an inverted Olympus IX71 microscope. Needles were made using borosilicate glass filaments with a 1.0 mm O.D. and 0.58 mm I.D. (BF100-58-10, Sutter Instruments) and a micropipette puller P-97 (Intracell). In all experiments except those displayed in Supplementary Fig. 2a , AcTEVTM Protease (Thermo Fisher, Cat. No. 12575) was used in a mix containing 10U/µl TEV protease in 50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 5 mM DTT, 50% (v/v) glycerol, 0.1% (w/v) Triton X-100. For Supplementary Fig. 2a we used TEV protease from GenScript (Cat. No. Z03030-1000) in a final mix containing 2.5 ng/µl TEV protease in 50 mM Tris, 5 mM DTT, 12.5% glycerol, pH 7.5. In indicated experiments 25 pmol of tetramethyl-rhodamine-5-dUTP (Roche) were added to the injection mix to evaluate the efficiency of germline microinjection by the incorporation of labelled nucleotides into the DNA of germ cells. Following microinjection, worms were rescued from the agarose pad with M9 salt buffer and placed in NG plates with Escherichiacoli OP50 for 3h30’ (unless otherwise indicated) before germline dissection.
4
SUMO3-SARS2-NP Fusion Protein Cleavage
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100 μM of SUMO3-SARS2-NP fusion protein with a TEV protease cut site in between SUMO3 and SARS2-NP was incubated with 0.2 IU/ml His-tagged TEV protease (GenScript) in buffer (25 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM DTT) at 4 °C overnight. Imidazole was then added into the protein solution at a final concentration of 10 mM. His-tagged TEV was removed using a column containing Ni-NTA. The cleavage result was analyzed by Coomassie-stained SDS-PAGE and stored at −80 °C.
5
Protein Purification and Tag Removal
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All of the protein samples were buffer exchanged into the phase separation buffer (25 mM Tris-HCl, pH 7.5) containing 150 mM NaCl unless otherwise noted. Prior to performing phase separation measurements, the His6-MBP-N10 tag was removed by the action of TEV protease (1:25 ratio) (GenScript USA Inc.) for 1 h at 30 °C. The completion of the cleavage reaction was judged by polyacrylamide gel electrophoresis (PAGE) and Coomassie blue staining.
6
Recombinant P8 Protein Purification
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A P8 gene with restriction enzyme sites (5ʹ-NdeI/3ʹ-SalI) was synthesized by Cosmogenetech (Seoul, Korea) and then cloned into the Escherichia coli expression vector (pET-22b; Novagen, Madison, WI, USA), which was described previously.27 The resultant pET-22b::P8 construct was transformed into Escherichia coli strain C41(DE3) (Novagen), which was cultured in M9 medium to an optical density (OD) of 0.6. The recombinant P8 protein (r-P8) contains a hexa-histidine (6×His) and a TEV protease cleavage site in its N-terminal region. R-P8 protein was purified by binding to Ni2+-NTA agarose (Qiagen, Valencia, CA, USA), and then the 6×His tag was removed using TEV protease (GenScript, Piscataway, NJ, USA). To confirm the homogeneity of the purified r-P8, r-P8 was applied to a size exclusion column (HiLoad 26/60 Superdex 200 pg; GE Healthcare, Boston, MA, USA).
7
Axoneme Immunofluorescence Imaging Protocol
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