Isopropyl β d 1 thiogalactopyranoside (iptg)

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Sourced in United States, China, Germany

IPTG (Isopropyl-β-D-1-thiogalactopyranoside) is a synthetic chemical compound used as an inducer in bacterial expression systems. It is a lactose analog that binds to the lac repressor, thereby deactivating it and allowing the transcription of genes under the control of the lac operon.

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Lab products found in correlation

Nickel nta agarose, by Qiagen (4 mentions) Dithiothreitol (dtt), by Avantor (3 mentions) Glycine, by Avantor (3 mentions) Sodium dodecyl sulfate (sds), by Avantor (3 mentions) Urea, by Merck Group (3 mentions) T4 dna ligase, by New England Biolabs (3 mentions) Phenyl methyl sulfonyl fluoride (pmsf), by Avantor (2 mentions) Bromophenol blue, by Bio-Rad (2 mentions) Bis acrylamide, by Merck Group (2 mentions) Tris buffer, by Avantor (2 mentions) L arginine, by Merck Group (2 mentions) Yeast extract, by BD (2 mentions) Ammonium persulfate, by Merck Group (2 mentions) Acrylamide, by Merck Group (2 mentions) Tryptone, by BD (2 mentions) Dna polymerase, by New England Biolabs (2 mentions) E coli bl21 de3, by Takara Bio (2 mentions) Dntps, by New England Biolabs (2 mentions) Pmd18 t vector, by Takara Bio (2 mentions) Restriction endonuclease, by New England Biolabs (2 mentions)

48 protocols using isopropyl β d 1 thiogalactopyranoside (iptg)

Genome-wide RNAi Screen in C. elegans

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The ORFeome RNAi library was used to conduct a genome-wide RNAi
screen⁴². For each day
of the RNAi screen, all bacterial colonies from two 96-well plates were cultured
for at least 12 h at 37°C in LB with 75 mg/L ampicillin. These cultures
were then pre-incubated with 1 mM IPTG (Amresco) for 1 h to maximize induction
of dsRNA production. 24-well plates with each well containing 2 mL NGM medium
with 1 mM IPTG (Amresco) and 75 mg/L ampicillin were prepared in advance and
stored at 4°C until needed; they were brought to room temperature a few
hours before seeding. Each bacterial colony culture was then seeded onto an
individual well of a 24-well plate and incubated for 24 h at 20°C. Three
L4 animals were picked into a 10 μl drop of M9 medium, which facilitated
their transfer into a well using a pipette. The progeny of these 3 animals were
screened 3 days later. Each set of RNAi clones screened also included a
pig-1 RNAi positive control and an empty pL4440 vector
negative control. The scorer was blinded to the identity of the RNAi clones.
Excretory cell counts were performed as described in Excretory cell counts below. Sanger sequencing was
used to confirm the identity of RNAi clones that reproducibly generated a Tex
phenotype for more than 10% of the animals scored.

Dwivedi V.K., Pardo-Pastor C., Droste R., Kong J.N., Tucker N., Denning D.P., Rosenblatt J, & Horvitz H.R. (2021). Replication stress promotes cell elimination by extrusion. Nature, 593(7860), 591-596.

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Screening and Characterization of scFv Phage

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For screening, 100 μl of supernatant containing rescued phage from each monoclonal (scFv) was added to the blocked streptavidin-coated plate coated with biotinylated preS1-peptide (5 μg/ml) and incubated for 1 hour at 37 ^oC. Post washing, the bound phage was detected using 100 μl of anti-M13 HRP-conjugated antibody (GE lifesciences, 27942101) (1:2000) using TMB as substrate. For further characterization, the phage containing supernatant was precipitated using PEG/NaCl and used in ELISA in a concentration of 1 × 10¹² phages/100 μl.
The scFv gene of selected scFvs was cloned into pAK400 vector and transformed into HB2151 E. coli cells for periplasmic expression. The scFv clone was then grown in 1L 2xYT/Chloramphenicol media and induced at an OD~0.8 with 1 mM IPTG (Amresco, 0487) for 15 hours at 20 °C, 250 rpm. The periplasmic extract was prepared using ‘Osmotic Shock method’60 (link). 100 μL of supernatant was added to the preS1-peptide coated streptavidin plate and incubated for 1 hour at 37 °C. Post washing, bound phages were detected using 100 μl of anti-his antibody (1:2000) (Cell Signaling Technologies, 3724) and anti-rabbit HRP-conjugated antibody (Cell Signalling Technologies, 70745) using TMB as substrate.

Sankhyan A., Sharma C., Dutta D., Sharma T., Chosdol K., Wakita T., Watashi K., Awasthi A., Acharya S.K., Khanna N., Tiwari A, & Sinha S. (2016). Inhibition of preS1-hepatocyte interaction by an array of recombinant human antibodies from naturally recovered individuals. Scientific Reports, 6, 21240.

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Purification and characterization of Cas13a

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Cas13a was purified as referred to in previous studies (East-Seletsky et al., 2016 (link), 2017 (link); Su et al., 2020 (link)). Briefly, the Cas13a gene in plasmid pC019-LwCas13a from Leptotrichia wadei (Addgene, United States) was subcloned into the psmarti vector (xhoI restriction site), and the correct plasmid was transformed into BL21(DE3) (General Biosystem, China). The expression of the target protein was induced at different temperatures (15 and 37°C) and different concentrations of IPTG (0.2 and 1.0 mM) (Amresco, United States). The Cas13a protein was purified by Ni-NTA (Smart-Lifesciences, China) using the 6 × His Tag antibody and horseradish peroxidase conjugate (Invitrogen, United States). After the addition of SUMO Protease (General Biosystem, China) to remove the fusion SUMO label, and the purified target protein was dialyzed into protein buffer [50 mM Tris, pH 7.5, 600 mM NaCl, 5% (vol/vol) glycerol, and 2 mM DTT].

An B., Zhang H., Su X., Guo Y., Wu T., Ge Y., Zhu F, & Cui L. (2021). Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification. Frontiers in Microbiology, 12, 732426.

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Recombinant Protein Expression and Purification

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Plasmids were transfected into E. coli BL21 (DE3) competent cells, and cultured at 37 °C. Protein expression was induced by adding 1 mM IPTG (AMRESCO, Cat# 0487) at OD₆₀₀ = 0.6–0.8. Cells were cultured at 16 °C overnight. The harvested cells were lysed by lysis buffer containing 150 mM NaCl (for hHSPA8, hHSPA8K71A, and hHSPA8-CT) or 300 mM NaCl (for RIP3_(388–518) in Fig. 4), 50 mM Tris-HCl, pH 8.0, 200 μg/mL PMSF and 0.2 mM β-mercaptoethanol, and sonicated. The supernatant was incubated with Ni-NTA agarose at 4 °C for 1 h, respectively. The Ni-NTA agarose was subsequently washed and then eluted with 500 mM imidazole. The eluted protein was further purified by gel filtration chromatography using the Superdex 200 10/300 GL column (GE Healthcare) at 4 °C in an ÄKTA FPLC system (GE Healthcare). Protein concentrations were determined by Bradford assay using bovine serum albumin (BSA) as standard.

Wu E., He W., Wu C., Chen Z., Zhou S., Wu X., Hu Z., Jia K., Pan J., Wang L., Qin J., Liu D., Lu J., Wang H., Li J., Wang S, & Sun L. (2023). HSPA8 acts as an amyloidase to suppress necroptosis by inhibiting and reversing functional amyloid formation. Cell Research, 33(11), 851-866.

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Purification of Rat Glucocorticoid Receptor

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¹⁵N-labelled wild-type and A477T mutant rat GR DBD (residues 440–525) were expressed and purified essentially as described previously13 (link) except that a codon optimized construct for expression in Escherichia coli was used here. In brief, proteins were expressed in E. coli (T7 Express; NEB) using the pET expression system in M9 minimal medium47 (link). Expression was induced at an OD₆₀₀ of 0.6–0.9 using 0.25 mM IPTG (Amresco). Temperature was lowered from 37 to 25 °C on addition of IPTG and cultures grown overnight. Cells were harvested and lysed followed by protein separation by IMAC and IEX chromatography. The latter was done after extensive dialysis against salt-free buffer. Final dialysis at the end of protein purification was carried out against NMR buffer (20 mM sodium phosphate; 100 mM NaCl; 1 mM DTT; pH 6.7).

Schöne S., Jurk M., Helabad M.B., Dror I., Lebars I., Kieffer B., Imhof P., Rohs R., Vingron M., Thomas-Chollier M, & Meijsing S.H. (2016). Sequences flanking the core-binding site modulate glucocorticoid receptor structure and activity. Nature Communications, 7, 12621.

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Purification of Recombinant Proteins from E. coli

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The Mdh was prepared according to a previously described method [14 (link)]. Briefly, E. coli strain harboring a recombinant plasmid constructed in a previous study [15 (link)] was grown overnight at 37° C in LB medium (Duchefa, Netherlands) and protein expression was induced by adding isopropyl β-d-1-thiogalactopyranoside (IPTG, Amresco, USA) at a final concentration of 0.3 mM. Then, the soluble protein fraction was purified using a His SpinTrap (GE Healthcare, UK) column according to the manufacturer's instructions after the pellet was sonicated at 10,000 Hz on ice. The proteins were constructed with a pCold vector containing a chaperone trigger factor (TF) to produce soluble and functional proteins. Therefore, as a vector control, TF was also purified using the above method. The endotoxin content of proteins was confirmed with a Pierce Chromogenic Endotoxin Quant kit (ThermoFisher, USA). The purity of proteins was confirmed using SDS-PAGE and Western blotting as previously described [15 (link)]. The concentration of purified proteins was measured by Pierce BCA protein assay kit (Pierce, USA) following the manufacturer’s instructions and stored at −20°C until use.

Shim S., Soh S.H., Im Y.B., Ahn C., Park H.T., Park H.E., Park W.B., Kim S, & Yoo H.S. (2020). Induction of systemic immunity through nasal-associated lymphoid tissue (NALT) of mice intranasally immunized with Brucella abortus malate dehydrogenase-loaded chitosan nanoparticles. PLoS ONE, 15(2), e0228463.

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Recombinant Mouse Tenascin-C Protein Expression

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Plasmids (pGEX-6P-1-mouse Tnc 2-700 or pGEX-6P-1-mouse Tnc 156-700) were transformed into BL21 (DE3) competent E. coli cells. 1 L cultures of BL21 each containing a different protein expression constructs were grown at 37 °C in LB and induced with 1 mM IPTG (AMRESCO, cat# 0487) at OD₆₀₀ = 0.4. Cells were incubated at 16 °C overnight and harvested. Cells were lysed in 80 mL lysis buffer (150 mM NaCl, 50 mM Tris/HCl pH 8.0, 1% glycerol, 200 μg/mL PMSF and 0.2 mM β-mercaptoethanol) and centrifuged. The cleared lysates were incubated with 4 mL glutathione-coupled Sepharose 4B beads (GE Healthcare, cat# 17-0756-01) at 4 °C overnight, and the beads were subsequently washed for three times then eluted with 30 mM GSH. The eluted proteins were loaded onto a Superose 6 Increase 10/300 GL (GE Healthcare, cat#29-0915-96) equilibrated in PBS/Az using an ÄKTA FPLC system at 0.3 mL/min and the A₂₈₀ of the eluate were monitored continuously. Mass standards used here were from a commercial HMW calibration kit (GE Healthcare).

Zhou S., Zhang W., Cai G., Ding Y., Wei C., Li S., Yang Y., Qin J., Liu D., Zhang H., Shao X., Wang J., Wang H., Yang W., Wang H., Chen S., Hu P, & Sun L. (2020). Myofiber necroptosis promotes muscle stem cell proliferation via releasing Tenascin-C during regeneration. Cell Research, 30(12), 1063-1077.

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Characterization of Hericium marmoreus Strain

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The H. marmoreus strain, Finc-W-247, was collected from Shanghai Finc Bio-tech Inc., Shanghai, China. H. marmoreus was sequenced with Pacbio and Illumina [43 (link)]. RNAiso Plus, PrimeScript^TM RT reagent Kit, and SYBR^® Premix Ex Taq II were purchased from TaKaRa (Beijing, China). TOP10 receptor cells were produced by Anhui Global Gene Company (Hefei, China). IPTG was purchased from Amresco (Beijing, China). LPS endotoxin ELISA kit was purchased from Solarbio (Beijing, China). Ni-NTA agarose column was purchased from Smart-Lifesciences (Changzhou, China). Modified Bradford Protein Assay kit was purchased from Sango (Shanghai, China). RAW 264.7 were obtained from the Chinese Academy of Sciences (Shanghai, China). DMEM medium was purchased from Gibco (Shanghai, China). Fetal bovine serum was purchased from Sigma (Port Melbourne, Australia). Penicillin-streptomycin and MTT were purchased from Beyotime (Shanghai, China). LPS was purchased from Sigma (St. Louis, MO, USA). IL-6, TNF-α, and IL-1β ELISA kits were purchased from Solarbio (Beijing, China). Rabbit anti-IL-6 polyclonal antibody, Rabbit anti-iNOS polyclonal antibody, and Rabbit anti-IL-1β polyclonal antibody were purchased from Bioss (Beijing, China).

Yu S., Wang Y., Wu Y., Bao D., Bing W., Li Y, & Chen H. (2023). Characterization, Recombinant Production, and Bioactivity of a Novel Immunomodulatory Protein from Hypsizygus marmoreus. Molecules, 28(12), 4796.

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In Vitro TFEB Acetylation Assay

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TFEB-His (WT or 4KR) was transformed into BL21 (DE3) and expression was induced with 0.5 mM IPTG (Amresco) at 37 °C. Bacterial cells were harvested and lysed by ultrasonication, and the proteins were analyzed by SDS-PAGE. Then, the target protein was purified by Ni-NTA beads 6FF (Bio-Rad). Flag-ELP3 protein was purified from HeLa cells 24 h after transfection by immunoprecipitation with anti-Flag affinity beads (Smart), then Flag-tagged protein was eluted with competitive elution buffer (50 mM Tris, 0.15 M NaCl, 200 μg Flag peptide (Smart)/ml, pH 7.4). For in vitro acetylation assay, recombinant TFEB protein was incubated with Flag-ELP3 immunoprecipitated from a cell lysate (TFEB KO cells) in the presence of acetyl-coA (1 mM) and 10 μl 5 × HAT assay buffer (250 mM Tris–HCl, pH 8.0, 5 mM DTT, 50% glycerol, 0.5 mM EDTA) in a total volume of 50 μl. The contents were gently mixed and placed in a 37 °C shaking incubator for 1 h. Then, protein loading buffer was added to the reaction and boiled for 5 min. The reaction products were subjected to SDS-PAGE and analyzed using immunoblotting.

Li T., Yin L., Kang X., Xue W., Wang N., Zhang J., Yuan P., Lin L, & Li Y. (2022). TFEB acetylation promotes lysosome biogenesis and ameliorates Alzheimer’s disease–relevant phenotypes in mice. The Journal of Biological Chemistry, 298(12), 102649.

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Purification and Characterization of E. coli Asparaginase

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Culture media ingredients, tryptone, and yeast extract were from Difco Laboratories, India. Tris buffer, glycine, IPTG, sodium dodecyl sulfate, PMSF, and deoxycholic acid were from Amresco, USA. Ammonium persulfate, acrylamide, bis-acrylamide, E. coli asparaginase, L-arginine and Urea were from Sigma Chemicals, USA. DEAE-sepaharose and S-200 gel filtration matrix was from GE Healthcare, Sweden. TEMED, EDTA, bromophenol blue from Biorad, USA. Coomassie brilliant blue R-250 and ampicillin from USB Corporation, Cleveland, Ohio. Glucose, NaCl, Nessler's reagent, and other chemicals were from Qualigen, India.

Upadhyay A.K., Singh A., Mukherjee K.J, & Panda A.K. (2014). Refolding and purification of recombinant L-asparaginase from inclusion bodies of E. coli into active tetrameric protein. Frontiers in Microbiology, 5, 486.

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