The yeast two-hybrid (Y2H) screen was performed as described previously (21 (link)). Briefly, human MCM6 was used as bait, and its potential E3 ligase was screened from a library containing ~400 open reading frames (ORFs) of human E3 ubiquitin ligases. The positive colony was able to survive in SD-4 medium (deficient in uracil, histidine, leucine and tryptophan) and could be stained with X-Gal (Sangon Biotech Co., Ltd.).
X gal
Manufactured by Sangon
Sourced in China
X-gal is a colorimetric substrate used in molecular biology for the detection and screening of β-galactosidase activity. It is a synthetic analog of lactose that produces a blue color when cleaved by the β-galactosidase enzyme.
Automatically generated - may contain errors
Lab products found in correlation
Isopropyl β d 1 thiogalactopyranoside (iptg), by Sangon (2 mentions)
Hygromycin h8080, by Solarbio (1 mentions)
Potassium ferricyanide, by Sangon (1 mentions)
Magnesium chloride, by Sangon (1 mentions)
Hoechst 33342, by Beyotime (1 mentions)
Dna gel extraction kit, by Sangon (1 mentions)
Peasy t1, by Transgene (1 mentions)
Transfast taq dna polymerase, by Transgene (1 mentions)
Smart race cdna amplification kit, by Takara Bio (1 mentions)
Tianscript rt kit, by Tiangen Biotech (1 mentions)
Total rna extraction kit, by Tiangen Biotech (1 mentions)
7 protocols using x gal
1
Yeast Two-Hybrid Screen for E3 Ligase
2
Screening and Verifying Recombinant Plasmids
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Phenotypes were used for initial screening to obtain the positive rates (positive colonies/total colonies). Cells that overexpress phbCAB formed white colonies on LB plates with 2% glucose due to the accumulation of polyhydroxybutyrate (PHB) (18 ). Colonies producing eGFP were checked for fluorescence by using FluorChem Q (ProteinSimple, USA). Blue-white screening was used to check for the expression of lacZ on LB plates with the appropriate antibiotic, 1 mM isopropyl β-d -1-Thiogalactoside (Sangon Biotech, China), and 0.5 mg/ml of X-gal (Sangon Biotech, China) (22 (link)). Ten to 20 positive colonies were then checked via colony PCR. Five or ten plasmids from the positive colonies were sent for sequencing at TsingKe Biological Technology Company (Beijing, China) to verify the inserts as indicated in the text. The PCR and sequencing primers for the plasmids used in TEDA optimization and testing were listed in Supplementary Table S2 .
3
Cultivation of Kluyveromyces Yeast Strains
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K. marxianus and K. lactis cells were cultivated at 30 degrees. YPD medium (2% peptone, 2% glucose, 1% yeast extract, 2% agar for plates). SD medium (0.17% yeast nitrogen base without amino acids and ammonium sulfate (LA7420, Solarbio, Beijing, China), 2% glucose, 0.1% glutamic sodium, 2 g/L dropout mix, 2% agar for plates) was prepared as described before [40 ]. YPD or SD medium was supplemented with 0.2 g/L G418 (A600958, Sango), 200 mg/mL Hygromycin (H8080, Solarbio, Beijing, China) or 80 mg/L X-gal (A600083, Sangon, Shanghai, China) as indicated.
4
Cellular Endocytosis Pathway Inhibitors
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Hoechst 33342 purchased from Beyotime (Shanghai, China). β-Galactosidase, potassium ferricyanide, potassium ferrocyanide, magnesium chloride and X-gal were obtained from Sangon Biotech (Shanghai, China). Chlorpromazine (CPZ, a clathrin-mediated endocytosis inhibitor [9 (link)], methylated-β-cyclodextrin (MβCD, a caveolae-mediated endocytosis inhibitor, a cholesterol depletion agent [10 (link)]), 5-(N-ethyl-N-isopropyl) amiloride (EIPA, a macropinocytosis inhibitor, selectively blocks the Na+/H+ antiporter [11 (link)]) and LY294002 (a macropinocytosis inhibitor, a strong phosphatidylinositol-3 kinase inhibitor [12 (link)]) were obtained from Aladdin Industrial (Shanghai, China).
5
Bacterial Quorum Sensing Biosensors
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AHL-producing bacteria were screened by using two AHL-sensing bacterial biosensors, Chromobacterium violaceum CV02613 (link) and Agrobacterium tumefaciens KYC55,14 (link) which respond to short-chain and long-chain AHLs, respectively, as described previously.15 (link),16 (link) Briefly, fresh cultures of QS biosensor strains were inoculated on LB agar plates. KYC55 was grown in LB medium supplemented with 100 μg/mL tetracycline and 100 μg/mL spectinomycin at 28 °C. CV026 was grown in LB medium supplemented with 20 μg/mL kanamycin at 28 °C. For both KYC55 and CV026, the agar was supplemented with 40 mg/mL X-gal (Sangon Biotech, Shanghai, China). Freshly cultured strains to be tested were streaked on the plates parallel to the biosensor strains. The plates were incubated overnight at 28 °C. Production of AHLs was indicated by the formation of the purple pigment, violacein, or by blue coloration due to β-galactosidase activity.9 (link),17 (link)
6
Cell-based Tetracysteine Tag Detection
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The TC-FlAsH In-Cell Tetracysteine Tag Detection Kit was purchased from Molecular Probes of Invitrogen, Inc. (Eugene, OR, USA). The onitrophenyl-β-galactoside (ONPG), isopropyl β-D-1-thiogalactopyranoside (IPTG), and X-gal were acquired from Sangon Biotech (Shanghai, China). The peptides of CPP-TBE and CPP-TBE-m used in this study are presented in Table S1. The peptides were synthesized at China Peptides Co., Ltd (Shanghai, China) and dissolved in ultrapure water to achieve a stock solution of 5 mM. Other reagents were purchased from Sinopharm Chemical Reagent (Shanghai, China). Phosphate-buffered saline (PBS) was prepared using 137 mM NaCl, 2.7 mM KCl, 10 mM Na 2 HPO 4 , and 1.8 mM KH 2 PO 4 . All the buffers were filtered through a 0.22 μm filter and used within 3 weeks.
7
Characterization of A. mongolicum Genes
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A. mongolicum seeds were provided by the Forage Experiment Station of Inner Mongolia University. Competent Escherichia coli cells Trans-T1, cloning vector PEASY-T1, and TransFast Taq DNA Polymerase were purchased from Transgen Biotech Company. A total RNA extraction kit and a TIANScript RT kit for first-strand synthesis were obtained from TIANGEN Biotech Co. Ltd. A SMART RACE cDNA Amplification Kit (Clontech) was purchased from TaKaRa Company. Other reagents, including a DNA gel extraction kit, IPTG, and X-gal were purchased from Sangon Biotech Co. Ltd. (Shanghai), and this company also conducted primer synthesis and sequencing.
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