Anti-OPN antibody was purchased from R&D Systems. Anti-Phospho H2AX (ser139, clone JBW301) and anti-ATM antibodies were purchased from Millipore. Anti-53BP1 was purchased from Novus Biologicals. Anti-Phospho ATM (ser1981), anti-Phospho 53BP1 (ser1778), anti-Chk2 and anti-Phospho Chk2 (thr68) antibodies were purchased from Cell Signalling. Anti-beta actin (clone AC-15) antibody was purchased from Sigma Aldrich. Anti-mouse Alexa 488 was purchased from Life Technologies. Secondary antibody rabbit anti-mouse HRP was purchased from Dako; secondary antibody donkey anti-goat HRP was purchased from Santa Cruz and secondary antibody goat anti-rabbit HRP was purchased from Invitrogen. IPTG (isopropyl β-D-1-thiogalactopyranoside) was purchased from Sigma Aldrich. Recombinant human osteopontin (full length) was a kind gift of Dr. Larry W. Fisher (National Institute of Dental and Craniofacial Research, Bethesda, MD, USA).
Isopropyl β d 1 thiogalactopyranoside iptg
Manufactured by Merck Group
Sourced in United States, China, Sao Tome and Principe, Germany
Isopropyl β-D-1-thiogalactopyranoside (IPTG) is a synthetic chemical compound commonly used in molecular biology and genetic engineering applications. It functions as a lactose analog that can induce the expression of genes under the control of the lac operon in bacterial cells.
Automatically generated - may contain errors
Lab products found in correlation
Kanamycin, by Merck Group (11 mentions)
Ampicillin, by Merck Group (9 mentions)
Sodium chloride (nacl), by Merck Group (7 mentions)
Imidazole, by Merck Group (6 mentions)
Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (6 mentions)
L arabinose, by Merck Group (5 mentions)
Lysozyme, by Merck Group (5 mentions)
5 bromo 4 chloro 3 indolyl β d galactopyranoside x gal, by Merck Group (5 mentions)
Ni sepharose 6 fast flow, by GE Healthcare (4 mentions)
T4 dna ligase, by New England Biolabs (4 mentions)
Chloramphenicol, by Merck Group (4 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (4 mentions)
Triton x 100, by Merck Group (3 mentions)
Sds page, by Thermo Fisher Scientific (3 mentions)
Tween 20, by Merck Group (3 mentions)
Tryptone, by Merck Group (3 mentions)
Restriction enzyme, by New England Biolabs (3 mentions)
Dithiothreitol (dtt), by Merck Group (3 mentions)
T4 dna ligase, by Thermo Fisher Scientific (3 mentions)
Glutathione sepharose 4b, by GE Healthcare (2 mentions)
176 protocols using isopropyl β d 1 thiogalactopyranoside iptg
1
Antibody Immunoblotting for DNA Damage Response
2
Reconstitution of Lipid-Protein Complexes
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1,2-Dioleoyl-sn-glycero-3-phospho-l-serine (PS), 1-palmitoyl-2-oleoyl-sn-glycero-3-phospho-(1′-rac-glycerol) (sodium salt) (PG), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (PE), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (PC), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(lissamine rhodamine B sulfonyl) (Rhod-PE), 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine-N-(7-nitro-2-1,3-benzoxadiazol-4-yl) (NBD-PE), and brain phosphatidylinositol 4,5-bisphosphate or PtdIns(4,5)P2 (PI[4,5]P2) were purchased from Avanti Polar Lipids; DDM (n-dodecyl β-D-maltoside) and OG (n-octyl glucoside) were from Gold Biotechnology; IPTG (isopropyl β-d-1-thiogalactopyranoside), Triton X-100, Hepes, KCl, imidazole, MβCD (methyl-β-cyclodextrin), and DSP (dithiobis succinimidyl propionate) were from Sigma-Aldrich; glutathione Sepharose 4 B and Ni Sepharose 6 Fast Flow were from GE Healthcare; β-mercaptoethanol and glycerol were from Thermo Fisher Scientific; Bio-beads SM2 were from BIO-RAD; BSA and protease inhibitor cocktail were from Roche; non-fat milk powder was from CST; Accudenz was from Axell; trypsin was from Thermo Fisher Scientific; cy3 and cy5 were from Cytiva. The antibodies used in this study are listed in Table S2.
Table S2
3
Production and Characterization of Anti-PLE Antibody
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Immunogen of PLE was prepared by conjugating the conserved sequences of PLE isoenzymes (SKEAAKKPPKIKC and CNTQAAKRLKGEE) to keyhole limpet hemocyanin, the rabbit derived PLE polyclonal antibody were prepared and purified as described previously48 (link). The cross-activity of the antibody was determined with 7 recombinant PLEs (PLE1 to PLE6, and APLE). The recombinant PLEs were expressed and purified as described by Böttcher et al.18 (link). In brief, the recombinant plasmid pET-15b-PLE constructed by our research group and molecular chaperone pGro7 were transfected into E. coli Origami (DE3), positive clones were selected to culture at 30 °C, 200 rpm. L-arabinose (Sigma) was firstly added to final concentration of 1 mg/mL to induce the expression of pGro7, when the optical density at 600 nm reached 0.6–0.8, IPTG (isopropyl β-D-1-thiogalactopyranoside) (Sigma) was added to final concentration of 40 µM to induce the expression of PLEs. After being cultured for 6 h at 30 °C, 200 rpm, the cells were collected by centrifugation (4 °C, 8000 rpm, 10 min) and broken. The samples were centrifuged for 30 min at 4 °C, 12000 rpm, and the supernatants were harvested and purified by AKTA purifier and His Trap FF crude column. The purified PLEs were analyzed by western blotting.
4
Cefotaxime Sodium Salt Antibiotic Protocol
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The solvent used to prepare and dilute antibiotic Cefotaxime sodium salt (CTX, Sigma Aldrich) was the M9 compound medium to avoid the concentration of the nutrients decreasing in the prepared culture medium. The original antibiotic stock solution (25 mg/mL CTX solution) was prepared by dissolving 66.49 mg Cefotaxime sodium salt (potency is 940 μg/mg) into 2.5 mL M9 compound medium (Andrews, 2001 (link)). Afterward, the original CTX solution was filtered (VWR, 0.2 μm cellulose acetate membrane) and further diluted to various gradient concentrations. In the following, 300 μL solution with different concentrations was transferred in 500 μL Eppendorf tubes with correlated concentration labels. All the prepared antibiotic stock solutions were kept in the freezer at −20°C for a maximum of 2 months. To enable the expression of all TEM motifs under the control of lacI repressor, 50 μM isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma Aldrich) was also added to the growth medium for each experiment (Gomes et al., 2020 (link)). The IPTG stock (5 mM) was prepared by dissolving 119.15 mg into a 100 mL M9 compound medium, followed by transferring 1 mL to 2 mL Eppendorf tubes and keeping it in the freezer at −20°C for a maximum of 2 months.
5
Ginsenoside Evaluation in Cell Assays
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Coumestrol (CS), 17β-estradiol (E2), dimethylsulfoxide, and isopropyl β-D-1-thiogalactopyranoside (IPTG) were purchased from Sigma-Aldrich (St. Louis, MO, USA) and TCI (Tokyo, Japan). Lipofectamine 2,000 transfection reagent, penicillin, and streptomycin were purchased from Invitrogen (Carlsbad, CA, USA). Dulbecco's modified Eagle's medium, and 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) were purchased from Gibco BRL (Grand Island, NY, USA). Fetal bovine serum was purchased from HyClone (Logan, UT, USA). Ginsenosides PPD(S, R) and PPT(S, R) were purchased from Yuanye Biotechnology Co., Ltd. (Shanghai, China). The structures of the ginsenosides are shown in Fig. 1 . All other reagents used were of analytical grade.
6
Recombinant Expression of Apx Toxin
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Plasmids encoding Apx toxin fragments were transformed into BL21 and BL21::ngtagt. Strains were cultured in LB broth, in an orbital shaker, supplemented with appropriate antibiotics to early exponential phase (OD600nm = 0.5) upon which expression was induced by the addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG, Sigma). Cultures were incubated for a further 16 h, sedimented by centrifugation and resuspended in 50 mM Tris HCl pH 8, 300 mM NaCl, 10 mM imidazole. Cells were lysed using BeadBug zirconium lysing tubes (Sigma) in a FastPrep homogeniser (MP Biomedicals) and insoluble material was removed by pelleting. Toxin fragments were subsequently purified from cell lysates by Ni-NTA affinity chromatography and eluted in 50 mM Tris HCl pH 8, 300 mM NaCl, 300 mM imidazole.
7
Recombinant Protein HcADRM Purification and Expression
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Recombinant plasmid expression HcADRM (pET-28a (+)/HcADRM; Uniprot: W6NKS2) was provided by Ministry of Education (MOE) joint international Research Laboratory of Animal Health and Food Safety, College of Veterinary Medicine, Nanjing Agriculture University (Nanjing, Jiangsu, China). Escherichia coli BL21 (Vazyme Biotech, Nanjing, Jiangsu, China) transformed with pET-28a (+)/HcADRM was induced, and the recombinant protein was purified as previously described [33 (link)]. Briefly, the recombinants were incubated at 37 °C until the optical density at 600 nm (OD600) of the culture reached 0.6. Isopropyl β-D-1-thiogalactopyranoside (IPTG; Sigma Aldrich, Shanghai, China) was added with the final concentration of 1 mM, followed by incubation for another 5 h. The expression of HcADRM was analyzed by 12% (w/v) SDS-PAGE. Recombinant protein was purified from the bacterial lysates using Ni2+ nitrilotriacetic acid column (GE Healthcare, Pittsburgh, PA, USA) according to the manufacturer’s instructions. The concentration of the purified rHcADRM was determined using a Bradford assay [34 (link)].
8
Optimizing PfCelTOS Expression
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To investigate the effect of codon usage on PfCelTOS expression, cultures were grown in the presence of 40 µg/mL kanamycin (Sigma Aldrich, St. Louis, MO) in 1 L Difco Terrific Broth (BD Biosciences, San Jose, CA) at 30 °C. Cells were induced by adding 0.1 mM isopropyl β-d -1-thiogalactopyranoside (IPTG) (Sigma Aldrich) at an OD600 ~ 0.8–1.0 for protein induction. Cell samples were collected every hour from the time of induction for 3 consecutive hours for analysis by SDS-PAGE (Invitrogen, Waltham, MA). Subunit fragments representing the N-terminus and C-terminus of PfCelTOS essentially were expressed using the same conditions as for full length PfCelTOS.
9
Expressing and Purifying Angiogenin Mutants
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The cDNA for human ANG gene (369 bp) was amplified by PCR from the plasmid pCMV6-XL4 (OriGene) and eventually cloned in the BamHI and EcoRI restriction sites of the E. coli expression vector pGEX-6P-2 (GE-Healthcare), with a C-terminal hexa-histidine His-tag. The point mutations -D22G and L35P- were generated in ANG through site directed mutagenesis (Stratagene). Protein expression was induced in E. coli BL21 cells by addition of 1 mM Isopropyl β-D-1-thiogalactopyranoside (IPTG) (Sigma), at an A600 of 0.6. Cells were harvested 4 hours post-induction, and cell pellets were disrupted by the addition of lysozyme (Sigma), followed by sonication (Branson sonifier 250, Netherlands). Following centrifugation at 10,000 rpm for 1 hour, the Angiogenin-GST fusion proteins were recovered in the soluble fraction. Purification of wild-type and mutated proteins was performed through standard Ni-nitrilotriacetic acid (Ni-NTA) affinity-based purification procedure (Qiagen). Fractions containing eluted fusion proteins were pooled, dialyzed and concentrated, before being further purified through size-exclusion chromatography using a Superdex 75 10/300 GL column (GE Healthcare). The concentration of wild-type and mutant proteins was determined using an extinction coefficient of 54945 M−1 cm−1 at 280 nm (http://web.expasy.org/protparam/ ).
10
Expression and Purification of hACE2, hTMPRSS2, and NRP1
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Human ACE2 (1-615aa) with C-terminal polyhistidine and human TMPRSS2 (1-492aa) with C-terminal polyhistidine were expressed in FreeStyle 293F cells (ThermoFisher) with polyethyleneimine (PEI; Sigma-Aldrich). Six days post transfection, the supernatant containing the secreted protein was harvested and syringe-filtered using a 0.45um filter. The filtered supernatant was then incubated with Ni-Sepharose beads (GE healthcare) for 2 h for binding. The beads bound with hACE2 or hTMPRSS2 were then washed and the protein was eluted with 20 mM Tris, 250 mM imidazole, 300 mM NaCl, and buffer were exchanged into 20 mM Tris- HCl, 100 mM NaCl, 2 mM b-mercaptoethanol and 15% glycerol, followed by concentration with Vivaspin 6 spin filters (Sartorius). The purified hACE2 and hTMPRSS2 were stored at −80°C until use.
NRP1-b1(273-427aa) with N terminal polyhistidine was expressed in BL21Star™-(DE3) cells (Invitrogen). Cells were grown at 37°C, and once the cells reached an O.D. value of 0.6, induction was done with 750uM isopropyl β-d -1-thiogalactopyranoside (IPTG) (Sigma). After induction, cells were grown at 22°C for 16 h, following which, they were harvested, centrifuged, lysed and purified using Ni-Sepharose fast flow beads as per stander means described above.
NRP1-b1(273-427aa) with N terminal polyhistidine was expressed in BL21Star™-(DE3) cells (Invitrogen). Cells were grown at 37°C, and once the cells reached an O.D. value of 0.6, induction was done with 750uM isopropyl β-
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