Immobilised GST-NHSL1-6, 7, 8 (mAb) or GST-aa387-546NHSL1 (pAb) (Fig. 1f ) were digested with Prescission protease (Amersham Pharmacia Biotech) and used to produce monoclonal antibodies by PEG induced fusion of primary B cells isolated from popliteal lymph nodes with the mouse myeloma cell line P3-X63-Ag-8. The NHSL1 monoclonal antibody was subcloned twice (clone C286F5E1; IgG1) and used for western blot: undiluted hybridoma supernatant, immunofluorescence: 50 μg/ml. GST-aa387-546NHSL1 (Fig. 1f ) after digestion with Prescission protease (Amersham Pharmacia Biotech) was used to raise polyclonal rabbit antiserum #4457 (Eurogentec) and used for western blot: 1:2000, immunofluorescence: 1:400. Commercial primary antibodies: EGFP, western blot: 1:2000 (11814460001, Roche); Myc, western blot: 1:5000 (M5546, 9E10, Sigma); MBP, western blot: 1:10,000 (E8032S, New England Biolabs); Abi1, western blot: 1:1000, immunofluorescence: 1:100 (MBL, clone 1B9, D147-3), Scar/WAVE1, immunofluorescence: 1:50 (BD 612276), Scar/WAVE2 rabbit mAb, western blot: 1:1000, immunofluorescence: 1:50 (D2C8, CST 3659), ARPC2, western blot: 1:1000, immunofluorescence: 1:100 (07-227-I-100UG, Millipore). Secondary antibodies: HRP-goat anti-rabbit, western blot: 1:2000 (P044801, Agilent-Dako); HRP-goat anti-mouse, western blot: 1:2000 (P044701, Agilent-Dako).
Prescission protease
Manufactured by Cytiva
Sourced in Japan
PreScission protease is a highly specific and effective enzyme used for the removal of affinity tags from recombinant proteins. It recognizes and cleaves the amino acid sequence Leu-Glu-Val-Leu-Phe-Gln↓Gly-Pro, allowing for the precise separation of the target protein from the affinity tag.
Automatically generated - may contain errors
Lab products found in correlation
Glutathione sepharose 4b, by Cytiva (3 mentions)
Complete protease inhibitor cocktail, by Roche (2 mentions)
Pgex 6p 1, by Cytiva (2 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (2 mentions)
Glutathione sepharose 4b column, by Cytiva (2 mentions)
Glutathione sepharose beads, by Cytiva (1 mentions)
Superdex 75 10 300 column, by Cytiva (1 mentions)
Limulus amebocyte lysate assay, by Lonza (1 mentions)
Opti mem 1, by Thermo Fisher Scientific (1 mentions)
Amicon filter, by Merck Group (1 mentions)
Hanks balanced salt solution (hbss), by Fujifilm (1 mentions)
Fetal bovine serum fbs, by Nichirei Biosciences (1 mentions)
Pei max, by Polysciences (1 mentions)
Dulbecco modified eagle medium (dmem), by Cytiva (1 mentions)
Dnase 1, by GE Healthcare (1 mentions)
Dnase 1, by Thermo Fisher Scientific (1 mentions)
Glutathione sepharose 4b, by GE Healthcare (1 mentions)
Hitrap q, by Cytiva (1 mentions)
Superdex 75, by Cytiva (1 mentions)
Histrap column, by Cytiva (1 mentions)
33 protocols using prescission protease
1
Monoclonal Antibody Production and Characterization
2
Expression and Purification of GST-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The Escherichia coli strain, DH5α, was transformed with a pGEX‐6P‐based plasmid encoding GST‐ERK2(K52N), GST‐MEK1(WT or D282N), or GST‐MEK2. Exponentially growing DH5α cells carrying one of these plasmids were incubated with 0.5 mm isopropylthio‐β‐galactosidase (IPTG) at 25 °C for 14 h before harvesting. DH5α cells carrying a pCold‐based plasmid encoding caspase‐3‐GST were incubated with 0.5 mm IPTG at 15 °C for 24 h. Cells were suspended in cold phosphate‐buffered saline, lysed by sonication, and then clarified by centrifugation (30 000 g for 15 min at 4 °C). The clear supernatant was filtered through a 0.45 μm filter, and GST‐tagged proteins were purified using glutathione sepharose beads (Cytiva, Marlborough, MA, USA) and used for in vitro assays. Where indicated, the GST‐tag of purified GST‐MEK1 and GST‐MEK2 was removed by incubating with PreScission Protease (Cytiva) at 4 °C for 24 h in buffer containing 50 mm Tris–HCl (pH 7), 150 mm NaCl, 1 mm EDTA, and 1 mm DTT.
3
Purification of mEGFP and EB1-GFP
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To obtain mEGFP, 0.5 mg of GFP-γ-TuNA (Wieczorek et al., 2020b (link)) was incubated with 0.5 mg of PreScission protease (Cytiva) on ice for 2 h. The reaction was gel-filtered over a Superdex 75 10/300 column (Cytiva) preequilibrated in 40 mM Hepes, pH 7.5, 150 mM NaCl, 1 mM MgCl2, and 2 mM 2-mercaptoethanol. Purified mEGFP was supplemented with glycerol to 10% (vol/vol), snap-frozen in liquid N2, and stored at −80°C. EB1-GFP in a pET-DUET vector (Novagen) was expressed and purified from BL21(DE3) Rosetta (Novagen) cells using Ni-NTA affinity followed by gel filtration using the methods described in Forth et al. (2014) (link).
4
Expression and Purification of Mouse ZO-1 PDZ1
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression and purification of the mouse ZO-1(PDZ1) (residues 18–110) were previously described [41 (link)]. In brief, the recombinant glutathione-S-transferase (GST)-tagged form of mouse ZO-1(PDZ1) was uniformly isotopically labeled with 15N by harvesting the recombinant Escherichia coli BL21(DE3) in minimal media with 15N-ammonium chloride as the sole nitrogen source. The cells were collected and disrupted, and the cell lysate was applied to GST-accept affinity resin (Nacalai Tesque, Kyoto, Japan) to capture the fusion protein. After on-column digestion by PreScission™ Protease (Cytiva, Tokyo, Japan), 15N-labeled ZO-1(PDZ1) was purified by gel filtration chromatography. ZO-1(PDZ1) was dissolved in 5% D2O–95% H2O containing 20 mM MES buffer (pH 5.9) for further NMR measurements.
5
Purification of Recombinant FimA Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The DNA plasmid vector PYT1245 expressing whole FimA protein was kindly provided by Dr. Yutaka Terao at Niigata University (18 (link)). Escherichia coli BL21 competent cells (BioDynamics Laboratory Inc., Tokyo, Japan) were transformed with PYT1245 by the heat-shock method and were cultured in Luria-Bertani medium supplemented with ampicillin (100 µg/ml). The supernatants from ultrasonicated E. coli BL21 transformants carrying the PYT1245 plasmid were applied to a GST affinity column (Cytiva, Sheffield, UK). The rFimA protein was eluted by cleaving the GST-rFimA fusion protein with PreScission protease™ (Cytiva, Sheffield, UK). The recovered protein was applied to the affinity column (JNC CORPORATION, Tokyo, Japan) to remove endotoxin, and the elution was employed as the purified rFimA protein. The Limulus amebocyte lysate assay (BioWhittaker, Walkersville, MD, USA) resulted in <0.1 endotoxin units of LPS per 1 µg of rFimA.
6
Rat cPLA2 C2 Domain Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The cDNA of rat cPLA2 C2 domain (residues 17-141; GenBank: NM_133551 ), with engineered Cys139Ala and Cys141Ser substitutions, was subcloned into the pGEX-6P-1 bacterial expression vector (Cytiva) using EcoRI and XhoI restriction sites. A polyhistidine tag was appended to the C-terminus of the C2 domain, followed by a unique cysteine for conjugation with thiol-reactive dyes or enzymes. This yielded an N-terminal glutathione S-transferase (GST) tag that could be cleaved by PreScission protease (Cytiva) and a C-terminal polyhistidine tag for sequential affinity chromatography. A second version of the probe, C2-XL, was made by inserting a (GlyGlySer)7 repeat between the polyhistidine tag and the unique cysteine. To generate mEos2-C2, the ORF of mEos2 was subcloned into pGEX-6P-1 using BamHI and EcoRI sites, flanked by N-terminal (GlyGlySer)6 and C-terminal (GlyGlySer)6 repeats. Subsequently, residues 17-141 of the C2 domain were subcloned into pGEX-6P-1-mEos2 using EcoRI and XhoI sites.
7
Reagents for Cell Culture and Protein Assays
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reagents for tissue culture and transfection, DMEM (#SH30002, Cytiva, Marlborough, MA, USA), fetal bovine serum (FBS) (#175012, NICHIREI, Tokyo, Japan), Opti-MEM1 (#22600-134, Gibco, Billings, MT, USA), PEImax (#24885-2, Polysciences Inc., Warrington, PA, USA), and sodium 4-phenylbutyrate (4-PBA) (#O0511, TCI, Tokyo, Japan) were purchased. For SDS-PAGE and immunoblotting, mouse anti-His monoclonal antibody (#652501, Biolegend, San Diego, CA, USA), rat anti-FLAG monoclonal antibody (#637301, Biolegend, USA), HRP-anti-O-GlcNAc (#12938, CST, Danvers, MA, USA), CBB-R-250 (#031-17922, FUJIFILM, Tokyo, Japan), cell lysis buffer (10X) (#9803, CST, USA), and cOmplete protease inhibitor cocktail (#11697498001, Roche, Indianapolis, IN, USA) were purchased. For protein purification, Ni-NTA agarose (#143-09763, FUJIFILM, Tokyo, Japan), PreScission protease (#27-0843-01, Cytiva, Marlborough, MA, USA), Amicon filter (#UFC901096, Millipore, Burlington, MA, USA), empty polyprep chromatography columns (#7311550, Bio-Rad, Hercules, CA, USA), HBSS (#084-08965, FUJIFILM, Tokyo, Japan), and imidazole (#095-00015, FUJIFILM, Tokyo, Japan) were purchased.
8
Purification of Recombinant Yeast Ubc4 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Recombinant yeast Ubc4 was purified essentially as described before17 (link) as GST-3C-Ubc4 from E. coli Rosetta2(DE3) harboring pGEX-UBC4 and was eluted as non-tagged Ubc4 by PreScission Protease (Cytiva Cat# GE27084301). E. coli cells were cultivated in 2 l of 2xYT medium with 100 µg/ml Ampicillin and 100 µg/ml Chloramphenicol at 37 °C until 0.7 of OD600. IPTG was added to the culture at final concentration of 0.1 mM and further incubated at 20 °C for 20 h. Cells were harvested by centrifugation and the cell pellet was frozen by liquid nitrogen. Cells were ground by mortar and pestle in liquid nitrogen. Resulting cell powder was lysed in lysis buffer (50 mM Tris-HCl pH 7.5, 300 mM NaCl, 5 mM MgCl2, 1 mM DTT, 0.5 mM PMSF, 0.01% NP-40, 10 unit/ml of DNase-I (Invitrogen Cat# 18047019)) and the lysate was cleared by centrifugation at 40,000 x g, 40 °C for 15 min in SS-34 rotor. Lysate of E. coli cells harboring pGEX-UBC4 was incubated with Glutathione Sepharose 4B (GE Healthcare Cat# 17-756-05) followed by wash with lysis buffer without MgCl2 and DNase-I for four times, and elution buffer (50 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM MgCl2, 1 mM DTT) for three times. Ubc4 was eluted from beads using 80 µl PreScissionProtease in 1 ml elution buffer at 4 °C for 16 h.
9
Purification of Histidine-Tagged SHMT Enzymes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The efmSHMT and ecSHMT constructs with N-terminal histidine tags and PreScission protease cleavage sites were expressed in E. coli BL21(DE3) cells. The proteins were chromatographically purified on a HisTrap column (Cytiva, Tokyo, Japan) and digested with PreScission protease (Cytiva). The solutions were then subjected to HiTrap Q (Cytiva) and Superdex 75 gel filtration chromatography steps (Cytiva). Each protein preparation was concentrated in 20 mM HEPES buffer (pH 7.5) containing 50 mM NaCl (final concentration, 15 mg/ml) and stored at 4 °C.
10
Expression and Purification of PAK4(300-591)
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A gene fragment encoding PAK4(300-591) was amplified using primers with BamHI and XhoI sites and cloned into a pGEX-6P1 vector. The L301A mutation was then generated by site directed mutagenesis. The protein was expressed in E. coli BL21(DE3)pLysS cells by inducing with 1.0 mM IPTG at OD600 = 0.4, grown overnight at 18 °C. The protein was purified on a GSTrap column (Cytiva) and the GST tag cleaved with PreScission protease (Cytiva). The protein was further purified using a 26/600 Superdex 75 pg column (Cytiva) in 50 mM Tris [pH 7.5], 150 mM NaCl, 10 mM DTT.
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!