A plasmid containing the human Cat S gene was obtained from Addgene (#11251) and subcloned into a pGEX 6P1 plasmid. Cysteine residues at positions 12, 25 and 110 or all three (12, 25 and 110) were mutated to serine using site directed mutagenesis (custom cloning core facility at Emory University, Atlanta, GA). Recombinant proteins were expressed in BL21-DE3 cells (Sigma-Aldrich, St. Louis, MO), purified using GST SpinTrap columns (GE Healthcare Life Sciences), and the GST tag was removed with PreScission Protease (GE Healthcare Life Sciences) according to the manufacturer’s instructions.
Gst spintrap column
Manufactured by GE Healthcare
Sourced in United Kingdom, Japan
The GST SpinTrap columns are designed for the purification of glutathione S-transferase (GST)-tagged proteins. These columns contain agarose beads coated with glutathione, which can selectively bind to GST-tagged proteins. The captured proteins can then be eluted from the column using a buffer containing free glutathione.
Automatically generated - may contain errors
Lab products found in correlation
Spintrap, by Cytiva (9 mentions)
Bl21 de3 cells, by Merck Group (1 mentions)
Prescission protease, by GE Healthcare (1 mentions)
Viafect, by Promega (1 mentions)
Complete protease inhibitor cocktail, by Merck Group (1 mentions)
Anti flag tag antibody, by Merck Group (1 mentions)
Odyssey infrared imager, by LI COR (1 mentions)
Ecori, by New England Biolabs (1 mentions)
Xhoi, by New England Biolabs (1 mentions)
Tnt coupled wheat germ extract system, by Promega (1 mentions)
X ray film, by Kodak (1 mentions)
T4 polynucleotide kinase, by New England Biolabs (1 mentions)
Mutanbest kit, by Takara Bio (1 mentions)
Pgex 4t 1, by Cytiva (1 mentions)
Slide a lyzer mini dialysis device, by Thermo Fisher Scientific (1 mentions)
Sypro ruby, by Thermo Fisher Scientific (1 mentions)
Complete protease inhibitors without edta, by Roche (1 mentions)
Pgex 6p 1 vector, by GE Healthcare (1 mentions)
Celllytic b, by Merck Group (1 mentions)
12 protocols using gst spintrap column
1
Recombinant Human Cathepsin S Mutants
2
Characterizing GST-IL-22R Interactions with STAT3
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To monitor the interaction between the different GST-IL-22R mutants generated previously27 (link) and STAT3, the proteins were expressed independently in COS-7 cells. Briefly, 4 × 105 COS-7 cells plated in a six-well plate were transiently transfected using ViaFect (Promega), according to the manufacturer’s recommendations. Two days later, cells were lysed in 500 µl of lysis buffer (1% Triton X-100, 10% glycerol, 10 mM Tris (pH 8), 150 mM sodium vanadate, 1 mM sodium fluoride, 5 mM EDTA, 1 mM DTT and cOmplete Protease Inhibitor Cocktail (Sigma)) and cell debris were removed by centrifugation. STAT3 was mixed with recombinant monobody at 10 µM for 8 h at 4 °C. Then, GST-proteins were added for an additional 16 h. Purification of GST was performed using GST SpinTrap columns (GE Healthcare Life Sciences) according to the manufacturer’s recommendations. Input and eluted samples were analyzed by western blot with an anti-STAT3 antibody (12640, CST). The membranes were then reprobed with anti-GST (RPN1236V, Sigma), anti-Flag tag antibody (F1804, Sigma) and anti-His tag antibody (12698, CST).
3
Purification and EMSA of ATFS-1
4
Generation of Staufen1–GST Fusion Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To generate Staufen1–glutathione S-transferases (GSTs) recombinant proteins, the hemagglutinin (HA)-tagged Staufen1 cDNA was PCR amplified from pcDNA3-RSV-Staufen1-HA (Wickham et al. 1999 (link)) with the primers described in Table 1 . The resulting PCR products were digested with EcoRI and XhoI (New England Biolabs) and cloned in the pGEX-4t-2 vector and transformed into E. coli BL21 cells. The colonies that contained the plasmid + insert were grown in LB broth, 0.1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to the bacterial culture to induce the expression of the GST fusion protein, and cells were solubilized with NP-40 lysis buffer (50 mM Tris–HCl pH 8.0, 150 mM NaCl, 0.5 mM EDTA, and 0.5% NP-40) 6 h after the addition of IPTG. These cell lysates were incubated in GST SpinTrap columns (GE Healthcare) for 30 min at room temperature. Columns were washed six times with TEN100 buffer (20 mM Tris pH 7.4, 0.1 mM EDTA, and 100 mM NaCl) to remove unbound proteins and subsequently incubated with 2 µg of recombinant NC protein for 2 h at 4°C. Captured complexes were washed three times with TEN100 buffer and elution was performed using elution buffer (50 mM Tris–HCl, pH8 and 10 mM glutathione). Samples were resolved by SDS-PAGE and probed using rabbit polyclonal antibodies against Staufen1 and NC by western blot analysis.
5
Recombinant Cdh1 and Borealin Protein Production
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
To prepare in vitro translated (IVT) Cdh1 recombinant protein, the TNT Coupled Wheat Germ Extract System (Promega) was used with pcDNA3.1-Cdh1 vector, following the manufacturer's protocol. For GST-tagged recombinant protein production, pGEX-4T-1-borealin vector was transformed to BL21(DE3) (Nippon gene) and GST-tagged borealin was expressed for 15 h at 18°C with 0.2 mM isopropyl-1-thio-D-galactopyranoside (IPTG). Cells were lysed using NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl pH 7.5, 1 mM PMSF, 5 mM benzamidine and 0.5% NP-40) and sonicated. After centrifugation, cleared lysate was collected and incubated in GST SpinTrap columns (GE healthcare) for 1 h at 4°C. Then, each column was washed three times with binding buffer (10 mM Tris-HCl pH 7.6, 150 mM NaCl, 0.5 mM EDTA, 10 mM NaF, 0.1% Nonidet P-40, 20 mM β-glycerophosphate and 20 nM okadic acid) and incubated with 15 μl IVT Cdh1 and binding buffer for 2 h at 4°C. After incubation, each column was washed five times with binding buffer and proteins were eluted using GST elution buffer (GE healthcare). The eluted proteins were analyzed using SDS–PAGE.
6
MYB Binding Site Mutational Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All five MYBBSs in the VdSTS promoter sequence (− 1400 to − 590) were mutated using the MutanBEST Kit (Takara, Dalian, China) to generate MYBBSm1–5 mutant fragments. To perform EMSAs, MYBBS and MYBBSm1–5 probe fragments were obtained by PCR amplification and labeled with [γ-32P] ATP using T4 polynucleotide kinase (New England Biolabs, Hitchin, UK). The VdMYB1 ORF was cloned into the pGEX-6P-1 vector, and transformed into Escherichia. coli strain BL21(DE3). IPTG was added to the bacterial culture to induce expression of the GST-VdMYB1 fusion protein. The expressed fusion proteins were purified using GST SpinTrap columns (GE Healthcare). Labeled probes were incubated with 50 ng of GST-VdMYB1 in binding buffer (10 mM Tris [pH 7.5], 5 mM MgCl2, 50 mM KCl, 100 μg/mL BSA, 2.5% glycerol, 1 mM DTT, and 50 ng/μL poly (dI-dC) for 25 min. The fusion protein-bound probes were separated from unbound probes using PAGE (5%). Gels were dried, and signal was detected by overnight exposure to X-ray film (Kodak).
7
Production and Validation of Anti-TolCV2 Antibody
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Rabbit polyclonal anti-TolCV2 antibody was produced as described in our previous study (Guo et al., 2020 (link)). The DNA fragment encoding tolCV2 was amplified from V. vulnificus MO6-24/O chromosomal DNA by PCR with the following primer pair (tolCV2-F-EcoRI: 5′-CGGAATTCATGGTTAACAAGCACCTATC-3′; tolCV2-R-XhoI: 5′-CCGCTCGAGTCATGAATGAAAAGCTCGG-3′). The resulting PCR products were then inserted into the expression vector PGEX-4T-1 (Amersham Pharmacia Biotech Inc., Piscataway, NJ) and the GST-TolCV2 fusion protein was purified by GST SpinTrap columns (GE Healthcare Life Science, Buckinghamshire, UK). The rabbit polyclonal anti-TolCV2 antibody was produced using New Zealand white rabbits, and the specificity of the polyclonal antibody against TolCV2 was confirmed by Western blotting.
8
Purification of Apicomplexan GNA1 Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Codon-optimized versions of apicomplexan GNA1 (PfGNA1, BbGNA1, CpGNA1, EtGNA1, TgGNA1, TaGNA1, and GnGNA1) native sequences were cloned in a pGEX 6P-1 vector containing an N-terminal glutathione S-transferase (GST) tag and transformed in E. coli BL21 (DE3). Cultures were grown for 16 h at 30 °C after induction, lysed and supernatants containing the glutathione S-transferase (GST)-tagged GNA1 protein were filtered and purified through GSTrap HP 1-ml or GST SpinTrap columns (GE Healthcare). The proteins of interest were then dialyzed (Thermo Scientific Slide-A-Lyzer MINI Dialysis Devices, 10 K MWCO) at 4 °C using Buffer A (50 mM Tris-HCl pH 7.5; 250 mM NaCl) and stored.
9
Recombinant VWF73 Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
VWF73 mutants were cloned into the pGEX6P-1 vector (GE Healthcare Life Sciences, Piscataway, NJ) with a COOH-terminal 6X-His tag and an NH3-terminal GST tag. Vectors were transformed into BL21 E. coli (Agilent, Santa Clara, CA), and peptide expression was induced for 2 hr with 1 mM IPTG upon reaching OD600nm of 0.6 in a shaker flask grown at 30°C. Bacteria were lysed using Cell Lytic B (Sigma) in the presence of Complete Protease Inhibitors without EDTA (Roche). Peptide purification was performed using sequential nickel-NTA agarose and glutathione sepharose columns as previously described [15 (link)]. Four mL nickel NTA agarose was equilibrated with wash buffer (20 mM Tris pH 7.4, 600 mM NaCl, 40 mM imidazole, and 0.01% tween 20) before passing induced cell lysates over the column. The column was washed with 10 column volumes of buffer before eluting with TBS containing 250 mM imidazole. Samples were concentrated and buffer exchanged into TBS using 10 kDa cut-off filter Microcon centrifuge concentrators (EMD Millipore, Billerica, MA). Concentrated samples were then applied to a GST Spin-Trap column (GE Healthcare Life Sciences), washed with TBS, and eluted with 1 mM L-glutathione. Peptides were concentrated and buffered exchanged into TBS, and purity was analyzed by SDS-PAGE followed by staining with SYPRO RUBY (Invitrogen). Proteins were stored at -80°C until use.
10
Pyk2 Phosphorylation Assay with Src Kinase
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
GST fusion proteins of Pyk2-WT and Pyk-Y402F were expressed from pGEX4T plasmids in BL21 DE3 E. coli strain and purified by using GST Spin Trap column (GE Healthcare Life Sciences) following manufacturer’s instructions. Purified GST-Pyk2 WT or Y402F were incubated with recombinant Src kinase (Millipore, Billerica, MA), and phosphorylated Pyk2 was examined by immunoblotting using anti-Pyk2-pY402 antibody.
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!