The largest database of trusted experimental protocols

G418 sulfate

Manufactured by AG Scientific
Sourced in United States

G418 sulfate is an antibiotic commonly used as a selection marker in cell culture experiments. It is effective against a wide range of eukaryotic cells, including mammalian, plant, and fungal cells. G418 sulfate functions by inhibiting protein synthesis, making it useful for selecting cells that have been successfully transfected or transduced with a resistance gene.

Automatically generated - may contain errors

7 protocols using G418 sulfate

Stable cell lines overexpressing GFP and the CD133‐GFP fusion protein were created by transfecting confluent PC3 or DU145 cells in 100 mm plates with 20 μg of pcDNA3.1/NT‐GFP::CD133 or pcDNA3.1/NT‐GFP plasmid using the FuGENE HD transfection reagent as described previously.18 Briefly, cells stably expressing GFP‐CD133 or GFP were selected using 0.4 mg/mL G‐418 sulfate (A.G. Scientific INC., San Diego, CA, USA) in DMEM containing 4 mM glutamine, 1 mM pyruvate, 4.5 g/L glucose, and 10% FBS. After transfection, surviving green fluorescent cells expressing various levels of CD133‐GFP or GFP were expanded into 60 mm wells. Stably transfected cells were further expanded and passaged onto 100 mm tissue culture plates using 0.05% trypsin/0.2 g/L EDTA/4Na in Hanks Balanced Salt Solution (Welgene) and cultured with 2 mg/mL G‐418 to maintain expression of GFP‐CD133 or GFP.
+ Open protocol
+ Expand
2

Construction and Characterization of BDH1 Deletion Mutant

All strains used in this study are described in Table 3. JHY617 strain, a BDH1 deletion mutant derived from JHY60514 (link), was generated by PCR-mediated homologous recombination. The bdh1Δ::KanMX6 cassette flanked by 300 bp upstream and 282 bp downstream of the BDH1 open reading frame was obtained by PCR amplification from genomic DNA of bdh1Δ strain (BY4741 bdh1Δ::KanMX6, EUROSCARF) as a template, using the primer pair of d_BDH1 F (5′-GATTTGCTCACGCTACTTTG-3′) and d_BDH1 R (5′-GCCATGCTTTGTTTTAGACG-3′). The resulting PCR product was transformed into JHY605 strain and transformants were selected on YPD plate (10 g/L yeast extract, 20 g/L bacto-peptone, and 20 g/L glucose) supplemented with 200 μg/mL G418 sulfate (AG Scientific, Inc.)
Yeast cells were cultured in YPD medium or in synthetic complete medium lacking histidine (SC-His) (20 or 50 g/L glucose, 6.7 g/L yeast nitrogen base without amino acids, and 1.92 g/L amino acids mixture lacking histidine).
+ Open protocol
+ Expand
FK replicon cells were infected with DUSP1 shRNA using the shRNA-lentiviral infection system (Sigma-Aldrich, St. Louis, MO). A negative control lentiviral particle (LV-cont) was also constructed. Cells in which DUSP1 expression was stably suppressed were established using shRNA-carrying lentivirus particles according to the method of Choi et al [18 (link)]. In brief, FK replicon cells were seeded at a density of 1 × 105 cells per well and infected with 5 multiplicity of infection (MOI) lentiviral particles in the presence of 8 μg/mL hexadimethrine bromide (Sigma-Aldrich) overnight. Stably infected cells were selected for 2 weeks using complete medium with 500 μg/mL G418 sulfate (A.G. Scientific, San Diego, CA) and 10 μg/mL puromycin (Sigma-Aldrich). Suppression of DUSP1 expression in selected cells was confirmed by relative quantitative real-time polymerase chain reaction (PCR) and Western blot analysis.
+ Open protocol
+ Expand
Nourseothricin was purchased from Werner Bioagents (Jena Germany). Hygromycin B was purchased from Gold Biotechnology (Goldbio.com, St. Louis, MO). G418 Sulfate (used to select for resistance to the KanR gene) was purchased from AG Scientific (San Diego, CA).
+ Open protocol
+ Expand
The over-expression construct of human ANGPTL4 (NM_139314) was created by PCR amplification of genomic DNA by Phusion® High-Fidelity DNA polymerase (Thermo Fisher Scientific), using the following primers (designed based on the GenBank Nucleotide Database of the NCBI website): ANGPTL4: S-5’-TCTCTCACCGGGTATGAGCGGTGCTCCGACGGCC-3’, AS-5’-GTGTCTTAATTAACTAGGAGGCTGCCTCTGCTGC-3’ The generated fragment was digested with AgeI and PacI and ligated into the corresponding sites of pQCXIP vector (Clontech Laboratories, Inc., Mountain View, CA, USA). PCR products of ANGPTL4 were sequenced and found to be identical to the published sequence. Production of infectious viruses and melanoma infection were performed as described previously [10 (link)].
To produce mCherry expressing cells, melanoma cells were similarly transduced with a pQCXIN-mCherry plasmid and were selected using 800μg/ml G418 Sulfate (A.G. Scientific, Inc., San Diego, CA, USA).
+ Open protocol
+ Expand
A model of OATP1B1‐expressing cells was created by transfecting HEK293 cells with the pIRES2‐EGFP vector (Clontech, Mountain View, CA) containing SLCO1B1 cDNA. Similarly, HEK293 cells were transfected with the pDream2.1/MCS vector (GenScript, Piscataway, NJ) containing Slco1b2 cDNA. The HEK293 (ATCC CRL1573) cell line was obtained from American Type Culture Collection (ATCC, Manassas, VA). This cell line was exclusively used to study drug transport, and was not authenticated by the authors. All stable cell lines were selected and maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% fetal bovine serum (FBS) and G418 sulfate (500–1,000 μg/mL; AG Scientific, San Diego, CA) at 37°C under 5% CO2.
Sorafenib and rifampin were obtained from Chemie Tek (Indianapolis, IN). General tritium‐labeled Sorafenib (specific activity, >1 Ci/mmol; radiochemical purity, >97.1%) was custom made by Moravek Biochemicals (Brea, CA), and [3H]estradiol‐17β‐D‐glucuronide (E2G; specific activity, 50.1 Ci/mmol; radiochemical purity, 99.0%), a positive control substrate for OATP1B1 and Oatp1b2, was obtained from American Radiolabeled Chemicals (St. Louis, MO).
+ Open protocol
+ Expand
Chang liver and T-REx-HeLa were purchased from ATCC (#CCL-13), and Invitrogen (R714-07), respectively. G418 sulfate was purchased from AG Scientific, doxycycline from BD Biosciences, and anti-MYPT1, anti-pMYPT1, anti-RhoA antibody were purchased from Cell Signaling. RhoA Pull-down Activation Assay Biochem kit was obtained from Cytoskeleton, and Alexa Fluor 488 goat anti-mouse IgG antibody, blasticidin, pcDNA6/TR vector and TRIZOL reagent were purchased from Invitrogen. Rhodamine phalloidin was obtained from Life Technologies, pSuperior.neo vector from OligoEngine, and Mo-MuLV reverse transcriptase from Promega. In Situ Cell Death Detection Kit was purchased from Roche. Annexin V-FITC Apoptosis Detection Kit, anti-α-tubulin antibody, blebbistatin, cycloheximide, 4′,6-Diamidino-2-phenylindole dihydrochloride (DAPI), HRP-conjugated goat anti-rabbit IgG antibody, HRP-conjugated goat anti-mouse IgG antibody, human tumor necrosis factor-α (hTNF-α), phosphatase inhibitor cocktail II, III, propidium iodide, protease inhibitor mixture, RNase A and Y-27632 were purchased from Sigma. DNAs were synthesized from Cosmogenetech (Korea). The His-tagged Tat-C3 transferase exoenzyme (pHis-Tat-C3) expression vector was provided by Jae Bong Park and the recombinant C3 transferase was prepared as previously described [20 (link)].
+ Open protocol
+ Expand

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

Sign up now

Revolutionizing how scientists
search and build protocols!