Competent E. coli BL21‐CodonPlus (DE3)‐RIPL strain were transformed with pEN‐GST‐SRSF5‐flag, pEN‐GST‐U1A‐His, pEN‐GST‐U1–70k‐His, pEN‐GST‐U1C‐His and empty vectors. Single colony of each construct was then grown in LB media at 37 °C until desired density, and then induced with 0.3 mm IPTG (Beyotime Biotechnology, #ST098) at 30 °C overnight. GST‐tagged proteins were purified using standard protocols with the GST‐tag Protein Purification Kit (Beyotime Biotechnology, #P2262) according to the manufacturer's instructions. For GST‐SRSF5‐flag, the GST‐tag was removed by 20 units of PreScission Protease (2 U µL−1; Beyotime Biotechnology, #P2302) at 4 °C overnight in 50 mm Tris‐HCl, 150 mm NaCl, 1 mm EDTA, 1 mm DTT, pH7.5. For GST pulldown, 300 ng of purified recombinant SRSF5‐Flag was incubated with 150 ng of glutathione bound GST‐tagged U1A‐His, U1‐10K‐His, U1‐C‐His or with negative control GST‐His in binding buffer (50 mm Tris, pH 7.5, 200 mm NaCl, 10% glycerol, 0.5% Triton‐X‐100, 10 mg mL−1 RNaseA), supplemented with 1× protease inhibitor complete EDTA‐free (Beyotime Biotechnology). The reaction volume was made upto 300 µL in total and incubated for 1 h at 4 °C. Beads were washed and bound proteins were resolved by SDS‐PAGE and analyzed by western blot.
Isopropyl β d 1 thiogalactopyranoside (iptg)
Manufactured by Beyotime
Sourced in China
IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a synthetic chemical compound commonly used in molecular biology and microbiology. It functions as an inducer, triggering the expression of specific genes in bacterial cells that contain the lac operon.
Automatically generated - may contain errors
Lab products found in correlation
Bca protein assay kit, by Beyotime (2 mentions)
Bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Prescission protease, by Beyotime (1 mentions)
Gst tag protein purification kit, by Beyotime (1 mentions)
Phanta super fidelity dna polymerase, by Vazyme (1 mentions)
X gal, by Beyotime (1 mentions)
Pucm t vector, by Beyotime (1 mentions)
Easytaq pcr supermix, by Vazyme (1 mentions)
Imidazole, by Beyotime (1 mentions)
Pet n his c his vector, by Beyotime (1 mentions)
Anti his beads, by Beyotime (1 mentions)
Freund s complete adjuvant, by Beyotime (1 mentions)
Incomplete freund s adjuvant, by Beyotime (1 mentions)
Beyomag anti gst magnetic beads, by Beyotime (1 mentions)
Glutathione sepharose bead, by GE Healthcare (1 mentions)
Beyogold gst tag purification resin, by Beyotime (1 mentions)
Kanamycin, by Beyotime (1 mentions)
Avanti j 26 xp, by Beckman Coulter (1 mentions)
Triton x 114, by Merck Group (1 mentions)
Ni nta his bind resin, by Qiagen (1 mentions)
15 protocols using isopropyl β d 1 thiogalactopyranoside (iptg)
1
Purification and Interaction of Spliceosomal Proteins
2
Antibiotic Standards Acquisition Protocol
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The standards of enrofloxacin, chloramphenicol, ampicillin, trimethoprim, and sulfamethoxazole were purchased from Dr. Ehrenstorfer (Germany). Ciprofloxacin standard was obtained from MedChemExpress (New Jersey, United States). Tetracycline standard was from China Institute of Veterinary Drug Control (Beijing, China), and 2 × EasyTaq PCR SuperMix and Phanta super-fidelity DNA Polymerase were purchased from Vazyme (Nanjing, China). The pUCm-T vector, IPTG, and X-Gal were from Beyotime Biotechnology (Nantong, China). The primers of this study were all synthesized by Genscript (Nanjing, China).
3
Recombinant TEKTIP1 Protein Purification and Antibody Generation
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Full-length mouse TEKTIP1 was cloned into the pET-N-His-C-His vector (Beyotime, Shanghai, China) and then transfected into the ER2566 E. coli strain (Weidi Biotechnology, Shanghai, China). Protein expression was induced by 1 mM IPTG (Beyotime) at 30 ℃ overnight. After centrifugation, the bacterial pellet was resuspended in buffer (50 mM Tris–HCl pH 8.0, 200 mM NaCl), and the proteins were released by sonication. After centrifugation, anti-His beads (Beyotime) were added to the supernatant and incubated overnight at 4 ℃. After washing, recombinant protein was eluted with 250 mM imidazole (Beyotime). Coomassie brilliant blue stain of the gel of purified TEKTIP1 was shown in Supplementary Fig. 6. Recombinant TEKTIP1 protein was emulsified at a 1:1 ratio (v/v) with Freund’s complete adjuvant (Beyotime) and administered subcutaneously into ICR female mice at multiple points. For the subsequent three immunizations, recombinant TEKTIP1 protein was emulsified with incomplete Freund’s adjuvant (Beyotime) at an interval of 2 weeks. One week after the last immunization, blood was collected, and the serum was separated.
4
Purification of Apoptosis-Related Proteins
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1 mM IPTG (Beyotime, #ST098-5g) was used to induce the expression of His-Caspase 9-4 M (E306A–D315A–D330A–C287A)-pET-30a, His-LC3B-pET-30a, His-BIRC6-N1 (aa 27–358) and His-BIRC6-N2 (aa 358–543) in BL21 for overnight at 16 °C. After incubation for 16 h, the cells were collected, washed once with ice-cold PBS. Lysis/binding /wash buffer contained 300 mM NaCl, 25 mM HEPES, 20 mM Imidazole, and 1 μg/mL Benzonase at pH 7.5. The resuspended cells were then lysed by sonication, and the lysate supernatant after centrifugation was incubated with Ni-NTA beads for 20 min at 4 °C. The His-tagged proteins were eluted by the elution buffer containing 300 mM NaCl, 25 mM HEPES, and 250 mM Imidazole, pH 7.5. The elute was further purified by gel filtration (Superdex TM 75 10/300 GL Columns, Cytiva) using buffer C (PBS supplemented with 100 mM KCl, pH 7.4). The fraction at 0.5 mL/tube was collected starting from 7 mL.
5
Purification and Analysis of GST and His-Tagged Proteins
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As previously described (31 (link)), cells were lysed with 1 × RIPA lysis buffer (P0013B, Beyotime) for 30 minutes at 4°C. Glutathione S-transferase (GST) fusion proteins were immobilized on BeyoMag Anti-GST Magnetic Beads (P2138, Beyotime). After washing with 1 × RIPA lysis buffer, the beads were incubated with cell lysates for 4 hours. The beads were then washed four times with 1 × RIPA lysis buffer and resuspended in loading buffer. The bound proteins were subjected to SDS/PAGE and Western blotting.
Escherichia coli BL21 was used to express GST-recombinant proteins and His-recombinant proteins after induction with IPTG (Beyotime). Then Escherichia coli BL21 was lysed with muramidase and sonication. Bacterial debris was removed by centrifugation for 10 minutes. Glutathione-Sepharose beads (GE Healthcare Life Sciences) were added to the liquid supernatant to purify GST-fusion proteins at 4°C overnight. The beads were then collected and washed for six times with binding buffer to remove the contaminating proteins. SDS-PAGE and high-sensitivity colloidal Coomassie blue staining were performed to examine the purification efficiency. For purification of His-tagged proteins, HisSep Ni-NTA MagBeads (Yeasen, 20561ES03) were used to isolate and purify His-tagged proteins. The remaining steps were similar to those for Glutathione-Sepharose beads.
Escherichia coli BL21 was used to express GST-recombinant proteins and His-recombinant proteins after induction with IPTG (Beyotime). Then Escherichia coli BL21 was lysed with muramidase and sonication. Bacterial debris was removed by centrifugation for 10 minutes. Glutathione-Sepharose beads (GE Healthcare Life Sciences) were added to the liquid supernatant to purify GST-fusion proteins at 4°C overnight. The beads were then collected and washed for six times with binding buffer to remove the contaminating proteins. SDS-PAGE and high-sensitivity colloidal Coomassie blue staining were performed to examine the purification efficiency. For purification of His-tagged proteins, HisSep Ni-NTA MagBeads (Yeasen, 20561ES03) were used to isolate and purify His-tagged proteins. The remaining steps were similar to those for Glutathione-Sepharose beads.
6
Recombinant Enzyme Expression and Purification
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The expression and purification of recombinant enzymes were conducted as our previous report [40 (link)]. Briefly, recombinant E. coli BL21(DE3)/pET24a(+) -csnA was induced with 0.5 mM IPTG (Beyotime Biotechnology, Shanghai, China) at 25 °C, 160 rpm for 60 h. The supernatants were harvested by centrifugation at 10000 × g for 20 min at 4 °C and further subjected to the Ni-Sepharose column. The purity and molecular weight of the protein were analyzed by SDS-PAGE. The protein concentration was detected using BCA protein assay kit (Beyotime Biotechnology, Shanghai, China). Enzyme activity was determined by 3,5-dinitrosalicylic acid (DNS) method.
7
GST-tagged Protein Purification and Interaction
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The recombinant plasmid pET-N-GST-p7 was transformed into E. coli BL21 (DE3) competent cells. The expression of GST or GST-p7 protein was induced by the addition of 1 mM IPTG (ST098, Beyotime). The bacterial cells were harvested and resuspended in cold PBS containing 1 mM PMSF, followed by mild sonication. GST or GST-p7 protein was purified with BeyoGoldTM GST-tag Purification Resin (P2250, Beyotime) according to the manufacturer’s instructions and then incubated with 200 μL of the lysates of HEK293T cells transfected with p3 × Flag plasmids for 2 h at 4°C. The resin was further washed five times with cold PBS, followed by protein detection by SDS-PAGE and immunoblotting.
8
Purification of His-tagged NCOA4 and GST-tagged Proteins
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N-terminal 6 × histidine-tagged NCOA4(383–522nd) or NCOA4 mutants were overexpressed in E. coli BL21 (DE3) Rosetta, induced by 0.2 mM IPTG (Beyotime) for 4 h at 37 °C. Cells were lysed by sonication in lysis buffer containing 50 mM NaH2PO4, 300 mM NaCl, 10 mM imidazole, pH 8. Nickel-nitrilotriacetic acid affinity columns (Smart-Lifesciences Biotechnology Co) were used for his-tagged NCOA4 protein purification.
The human FTH, NCOA4 (383–522nd), and HERC2 (2540–2700th) proteins were expressed as a GST N-terminal fusion protein in E. coli BL21 (DE3) Arctic strain. The GST-FTH fusion protein was induced with 0.2 mM IPTG for 16 h at 16 °C. The GST-NCOA4 (383–522nd) and -HERC2 were induced with 0.2 mM IPTG for 4 h at 37 °C. Cells were harvested by centrifugation, resuspended in PBS pH 7.4 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3), and lysed by sonication. The purification approach follows the manufacturer’s guidance (GE Healthcare).
The human FTH, NCOA4 (383–522nd), and HERC2 (2540–2700th) proteins were expressed as a GST N-terminal fusion protein in E. coli BL21 (DE3) Arctic strain. The GST-FTH fusion protein was induced with 0.2 mM IPTG for 16 h at 16 °C. The GST-NCOA4 (383–522nd) and -HERC2 were induced with 0.2 mM IPTG for 4 h at 37 °C. Cells were harvested by centrifugation, resuspended in PBS pH 7.4 (140 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.3), and lysed by sonication. The purification approach follows the manufacturer’s guidance (GE Healthcare).
9
Generation and Characterization of PDZK1 and EGFR Plasmids
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PDZK1 overexpression and knockdown plasmids were a kind gift from Dr. Beard (University of Adelaide) [46 (link)]. pBK-CMV-Flag-EGFR plasmid was kindly provided by Dr. Rockman. pcDNA3.1-PDZK1-Flag PDZ domain mutants (MTs, YGF in PDZ domain was mutated to AGA), pBK-CMV-Flag-EGFR-MT (key residues L1042/1063 of PDZ binding motif were mutated to F) and pcDNA3.1-Myc-c-Cbl plasmid were from Zeqiong Biotechnology Co., Ltd (Changsha, China).
The Glutathione S-transferase (GST)-tagged EGFR-CT (aa 1022–1186) wild type (GST-EGFR-CT-WT), EGFR-CT-MT and full length PDZK1, His-tagged PDZK1 PDZ1–4 plasmids were generated via PCR amplification, then inserted into pGEX-4T-1 and pET-28a, respectively and verified by DNA sequencing. IPTG (Beyotime, Shanghai) was used to induce the expression of fusion proteins.
The Glutathione S-transferase (GST)-tagged EGFR-CT (aa 1022–1186) wild type (GST-EGFR-CT-WT), EGFR-CT-MT and full length PDZK1, His-tagged PDZK1 PDZ1–4 plasmids were generated via PCR amplification, then inserted into pGEX-4T-1 and pET-28a, respectively and verified by DNA sequencing. IPTG (Beyotime, Shanghai) was used to induce the expression of fusion proteins.
10
Recombinant hGM-CSF Protein Expression
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The plasmid PET24a(+)-SA-hGM-CSF was transformed into the BL21(DE3) Competent cells. 5μL BL21(DE3)-PET24a-SA-hGM-CSF bacterial liquid was injected into the 10 ml LB liquid medium containing 50 μg/mL kanamycin (Beyotime), then the LB liquid medium was incubated overnight at 37°C with shaking on the shaker (IKA KS 4000 I control, Germany) and next inoculated into 500 mL culture medium containing kanamycin in the following night. It was inoculated into a 10L fermentor (NBS, BIOFLO 415, USA) and fermented in a ratio of 1:20 at OD600 = 1.5 . Through the previous exploration of the induction conditions, we found that it is best to add IPTG (Beyotime) to a final concentration of 0.5 mM at OD600 = 3 and continued for 10 hours. Finally, the cells were harvested by centrifugation at 4000 rpm at 4°C for 15 minutes (Beckman Coulter, Avanti j-26 XP, USA).
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