The ssDNA oligonucleotides and SARS-CoV-2 nucleocapsid protein coding sequences used in this study were synthesized by Beijing Aoke Dingsheng Biotechnology Co., Ltd. The specific sequences of the ssDNA oligonucleotides are provided in Table S1. The following reagents and kits were purchased from the respective suppliers: isopropyl β-D-thiogalactoside (IPTG), kanamycin, Lipo293 Transfection Reagent, Bradford Protein Concentration Determination Kit, the His-tag Purification Resin, Alexa Fluor 555-labeled Donkey Anti-Rabbit (H + L) antibody, Anti-Flag Magnetic Beads, and Anti-His Magnetic Beads were obtained from Beyotime Co., Ltd. Taq DNA polymerase, T4 DNA ligase, and CCK-8 Cell Counting Kit were purchased from Vazyme Biotechnology Co., Ltd. Anti-Flag Monoclonal Antibody, Anti-His Monoclonal Antibody, and anti-GAPDH Monoclonal Antibody were purchased from Proteintech Group, Inc. Dulbecco’s modified Eagle’s medium (DMEM; high glucose) and penicillin-streptomycin (liquid) were obtained from Thermo Fisher Scientific, Inc.
Kanamycin
Manufactured by Beyotime
Sourced in China
Kanamycin is a broad-spectrum antibiotic that inhibits the growth of various bacteria by interfering with protein synthesis. It is commonly used in molecular biology and microbiology applications as a selective agent in bacterial cultures.
Automatically generated - may contain errors
Lab products found in correlation
Penicillin streptomycin liquid, by Thermo Fisher Scientific (1 mentions)
Anti his monoclonal antibody, by Proteintech (1 mentions)
Anti his magnetic beads, by Beyotime (1 mentions)
Anti flag monoclonal antibody, by Proteintech (1 mentions)
Taq dna polymerase, by Vazyme (1 mentions)
His tag purification resin, by Beyotime (1 mentions)
Anti flag magnetic beads, by Beyotime (1 mentions)
Lipo293 transfection reagent, by Beyotime (1 mentions)
Isopropyl β d thiogalactoside, by Beyotime (1 mentions)
Anti gapdh monoclonal antibody, by Proteintech (1 mentions)
T4 dna ligase, by Vazyme (1 mentions)
Cck 8 cell counting kit, by Vazyme (1 mentions)
Bradford protein concentration determination kit, by Beyotime (1 mentions)
Imidazole, by Merck Group (1 mentions)
Isopropyl β d thiogalactopyranoside iptg, by Merck Group (1 mentions)
Escherichia coli strain bl21 de3, by Transgene (1 mentions)
Glycerol, by Merck Group (1 mentions)
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (1 mentions)
H1299, by Thermo Fisher Scientific (1 mentions)
Hek293t cell line, by Thermo Fisher Scientific (1 mentions)
7 protocols using kanamycin
1
Characterization of SARS-CoV-2 Nucleocapsid Protein
2
Protein Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression and purification were done using Escherichia coli strain BL21 (DE3; Transgene). Transformed E. coli BL21 clones were grown at 37 °C in LB medium with 50 μg/ml Kanamycin (Beyotime) to OD600 of 0.6–0.8. Isopropyl-β-d -thiogalactopyranoside (IPTG; Sigma Aldrich) was added to a final concentration of 0.4 mM to induce protein expression at 16 °C for 16 h. Bacteria were lysed in lysis buffer (20 mM Tris pH 7.8, 500 mM NaCl, 5% glycerol (Sigma Aldrich), 3 mM β-ME (Sigma Aldrich), and 10 mM imidazole (Sigma Aldrich)) with sonication, and centrifuged at 18,000×g for 45 min at 4 °C. The supernatant was purified using Ni-IDA beads resin (ProbeGene). Briefly, the resin was equilibrated with buffer A (20 mM Tris, pH 7.8, 500 mM NaCl, 5% w/v glycerol, and 10 mM imidazole). The supernatant was incubated with Ni-IDA beads at 4 °C for 30 min with rotation. After centrifugation, the resin was the washed with buffer A for three times, and eluted with buffer B (buffer A with 300 mM imidazole). Protein was desalted and concentrated with a 30 K molecular weight cutoff (MWCO) protein concentrator (Pierce), quantified and aliquoted.
3
Cell Culture and Reagent Conditions
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293T, H1299, and A549 cell lines were obtained from the American Type Culture Collection, and they had been verified. HEK293T cell line was cultured in Dulbecco's modified Eagle's medium (Gibco; C11995500BT) supplemented with 10% fetal bovine serum (FBS) (Hyclone; SV30087.03). A549 cell line was cultured in Dulbecco's modified Eagle's medium/F12 = 1:1 (Gibco; C11330500BT) supplemented with 10% FBS. H1299 cell line was cultured in RPMI1640 (Gibco; C11875500BT) supplemented with 10% FBS. All cells were cultured at 37 °C with 5% carbon dioxide.
Reagents: IPTG (Sangon Biotech; A100487-0005), Kanamycin (Beyotime; A506636-0025), MG132 (MCE; HY-13259), TM (Beyotime; SC0393-10 mM), ATP (Sangon Biotech; A600020), GSK2606414 (MCE; HY-18072), and CHX (MCE; HY-12320).
Reagents: IPTG (Sangon Biotech; A100487-0005), Kanamycin (Beyotime; A506636-0025), MG132 (MCE; HY-13259), TM (Beyotime; SC0393-10 mM), ATP (Sangon Biotech; A600020), GSK2606414 (MCE; HY-18072), and CHX (MCE; HY-12320).
4
Antimicrobial Activity Evaluation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Four types of bacteria and fungi, namely, Staphylococcus aureus, Escherichia coli, Pseudomonas aeruginosa, and Candida albicans (ATCC, Manassas, VA, USA), were cultured under standard conditions. Candida albicans was cultured with Sabouraud glucose broth medium (Solarbio Life Science, Beijing, China) at 30 °C and 200 rpm; Staphylococcus aureus, Escherichia coli, and Pseudomonas aeruginosa were cultured with Luria Broth (LB) liquid medium (Solarbio Life Science, Beijing, China) at 37 °C and 200 rpm. The paper disks containing 30 μg Kanamycin (Beyotime Biotechnology, Shanghai, China) and 30 μg Amphotericin (Beyotime Biotechnology) were assigned as the positive control, and the medium was assigned as the negative control. The paper disks incorporated with 30 μg of AMP were deposited on the surface of the agar plates pre-inoculated with the bacterial strain to be tested. Specifically, 60 μg AMPs were used to test against Candida albicans. The agar plates were incubated for 24 h under appropriate conditions. Candida albicans was cultured in Sabouraud glucose agar medium in an inverted incubator at 30 °C for 12 h, and the remainder was cultured in LB solid medium in an inverted incubator at 37 °C for 12 h. The diameter of the inhibition zone was measured for statistical analysis. Each test was performed in triplicates.
5
Recombinant hGM-CSF Protein Expression
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
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).
6
Cloning and Sequencing Recombinant Plasmids
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Competent Escherichia coli DH5α cells were grown on lysis buffer medium plates containing kanamycin (Beyotime Institute of Biotechnology) at 37°C for 18 h. Six colonies were selected and used to inoculate the kanamycin-containing LB liquid medium (50 μg/ml), which was agitated using a blender (Beijing Tianshi Tianli Medical Device Technology Development Center, Beijing, China) overnight. The plasmids were extracted by alkaline lysis and digested sequentially with BamHI and PstI. The positive recombinant vectors were digested with BamHI, but not PstI. Two clones were selected for each vector and sequenced by Invitrogen Applied Biosystems (Shanghai, China).
7
Recombinant Protein Purification and Refolding
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The plasmids pET-32a/edBjtshr, pET-32a/edDrtshr, pET-28a/Bjgpa2, pET-28a/Drtsha, pET-28a/Bjgpb5, and pET-28a/ Drtshb were transformed into Escherichia coli Transetta (DE3), and the cells were cultured overnight in lysogeny broth containing 100 mg/mL of ampicillin (Beyotime) (for pET-32a/ edBjtshr and pET-32a/edDrtshr) or 50 mg/mL of kanamycin (Beyotime) (for pET-28a/Bjgpa2, pET-28a/Drtsha, pET-28a/ Bjgpb5, and pET-28a/Drtshb). When OD 600 reached ;1.0, isopropyl b-D-thiogalactoside (IPTG) (Amresco, West Grove, PA) was added to the cultures at a final concentration of 1 mM, and the cultures were allowed to shake for 12 hours at 28°C. The recombinant proteins that were expressed in inclusion bodies were purified and refolded according to the method of Xu and Zhang (31) with modification of dialysis pH value. The refolded proteins were analyzed by SDS-PAGE on 12% gel and immunostained using mouse anti-His-tag antibody (32) as the primary antibody, followed by incubation with horseradish peroxidase-conjugated goat anti-mouse IgG (33) according to the method described previously by Wang et al. (34) . Protein concentrations were determined by the bicinchoninic acid methods using BSA as a standard (CWBIO, Beijing, China).
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!