Cells were incubated for 24 h in complete media supplemented with 1 µg/ml doxycycline and 50 µM biotin. After three PBS washes, cells (for small-scale analysis, <107; for large scale analysis, 4 × 107) were lysed at 25°C in 1 ml lysis buffer (50 mM Tris, pH 7.4, 500 mM NaCl, 0.4% SDS, 5 mM EDTA, 1 mM DTT, and 1x Complete protease inhibitor [Roche]) and sonicated. Triton X-100 was added to 2% final concentration. After further sonication, an equal volume of 4°C 50 mM Tris (pH 7.4) was added before additional sonication (subsequent steps at 4°C) and centrifugation at 16,000 relative centrifugal force. Supernatants were incubated with 600 µl Dynabeads (MyOne Steptavadin C1; Invitrogen) overnight. Beads were collected and washed twice for 8 min at 25°C (all subsequent steps at 25°C) in 1 ml wash buffer 1 (2% SDS in dH2O). This was repeated once with wash buffer 2 (0.1% deoxycholate, 1% Triton X-100, 500 mM NaCl, 1 mM EDTA, and 50 mM Hepes, pH 7.5), once with wash buffer 3 (250 mM LiCl, 0.5% NP-40, 0.5% deoxycholate, 1 mM EDTA, and 10 mM Tris, pH 8.1) and twice with wash buffer 4 (50 mM Tris, pH 7.4, and 50 mM NaCl). 10% of the sample was reserved for Western blot analysis. Bound proteins were removed from the magnetic beads with 50 µl of Laemmli SDS-sample buffer saturated with biotin at 98°C. For the larger scale preparation, 90% of the sample to be analyzed by mass spectrometry was washed twice in 50 mM NH4HCO3.
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Doxycycline
Doxycycline
Doxycycline is a broad-spectrum tetracycline antibiotic used to treat a variety of bacterial infections.
It is effective against both Gram-positive and Gram-negative bacteria, including Chlamydia, Mycoplasma, Rickettsia, and some Protozoa.
Doxycycline is commonly prescribed for respiratory tract infections, skin and soft tissue infections, sexually transmitted diseases, and other conditions.
It works by inhibiting bacterial protein synthesis, preventing the bacteria from growing and reproducing.
Doxycyclne is generally well-tolerated, but can cause side effects such as gastrointestinal upset, photosensitivity, and teeth discoloration in children.
Researchers can utilize PubCompare.ai to optimize doxycycline protocols through AI-driven comparisons of literature, preprints, and patents, helping to identify the most reproducible and accurate approaches and acheiving better results.
It is effective against both Gram-positive and Gram-negative bacteria, including Chlamydia, Mycoplasma, Rickettsia, and some Protozoa.
Doxycycline is commonly prescribed for respiratory tract infections, skin and soft tissue infections, sexually transmitted diseases, and other conditions.
It works by inhibiting bacterial protein synthesis, preventing the bacteria from growing and reproducing.
Doxycyclne is generally well-tolerated, but can cause side effects such as gastrointestinal upset, photosensitivity, and teeth discoloration in children.
Researchers can utilize PubCompare.ai to optimize doxycycline protocols through AI-driven comparisons of literature, preprints, and patents, helping to identify the most reproducible and accurate approaches and acheiving better results.
Most cited protocols related to «Doxycycline»
Biotin
Buffers
Cells
Centrifugation
Deoxycholate
Doxycycline
Edetic Acid
HEPES
Laemmli buffer
Mass Spectrometry
Nonidet P-40
Protease Inhibitors
Proteins
Sodium Chloride
Triton X-100
Tromethamine
Western Blot
Cells were transduced at a MOI between 0.5 and 1. Viruses and cells were incubated overnight in D-MEM media (Invitrogen) containing 6 µg/ml of polybrene (Sigma) in a final volume of 950 µl for 6-well plates, 5 ml for 10 cm dishes and 11 ml for 15 cm dishes. The next day, the viruses were removed, the cells were rinsed twice with PBS and fresh media was added. For primary cells, a second round of transduction was done. Drug selection was added at 48 h post-transduction. The following concentrations were used: blasticidin: 2.5 µg/ml for WI38, HCA2, BJ cells and 5 µg/ml for the other cells, hygromycin: 100 µg/ml for WI38, HCA2, BJ cells, 200 µg/ml for U2OS cells and 300 µg/ml for HT1080 cells, neomycin: 300 µg/ml for WI38, HCA2, BJ cells and 800 µg/ml for the other cells, puromycin: 0.5 µg/ml for HeLa cells and 2.0 µg/ml for other cells, zeocin: 400 µg/ml for HT1080 cells and 200 µg/ml for other cells. Induction of the cDNA/shRNA/miRNA was typically done by addition of doxycycline at a final concentration of 1.0 µg/ml for 48 h (cDNA) or 96 h (shRNA/miRNA) unless indicated otherwise.
Cells
DNA, Complementary
Doxycycline
HeLa Cells
hygromycin A
Hyperostosis, Diffuse Idiopathic Skeletal
MicroRNAs
Neomycin
Pharmaceutical Preparations
Polybrene
Puromycin
Short Hairpin RNA
Virus
Zeocin
Cloning Vectors
Culture Media
Dissection
DNA, Complementary
Doxycycline
Embryo
Fibroblasts
Genes
Heterozygote
Homozygote
Microscopy
Mus
Tail
Cells
Doxycycline
Feeder Cell Layers
Fibroblasts
Gelatins
Homo sapiens
Human Embryonic Stem Cells
Human Induced Pluripotent Stem Cells
Hyperostosis, Diffuse Idiopathic Skeletal
Induced Pluripotent Stem Cells
Infection
Lentivirus
Polybrene
Transfection
Trypsin
Virus
antibiotic G 418
Antibiotics
Cells
Culture Media
Doxycycline
Germ Cells
hygromycin A
Hyperostosis, Diffuse Idiopathic Skeletal
Polybrene
Puromycin
Retroviridae
Viral Genome
Virus
Most recents protocols related to «Doxycycline»
Example 2
The DNA encoding the amino acid sequence of human KIF5B-RET variant 1 was placed in a lentivirus vector under a doxycycline-inducible promoter to maximize expression with a carboxyl-terminal FLAG epitope to facilitate immunodetection of the fusion by anti-FLAG antibodies. Lentiviral-mediated gene transduction was used to express KIF5B-RET in Ba/F3 cells, KIF5B-RET dependent cells were selected by IL-3 withdrawal and confirmed to express the KIF5B-RET fusion protein by immunoblot analysis. To generate Ba/F3 cells carrying V804 substitutions, WT KIF5B-RET Ba/F3 cells were mutagenized overnight with ENU and plated in 96-well plates for a period of 2 weeks in the presence of 6 concentrations of MKIs (ponatinib, regorafenib, cabozantinib, or vandetanib). The concentrations chosen ranged from 2×-64× the proliferation IC50 for each compound: 125 nM to 4 μmol/L cabozantinib, 20 to 640 nM ponatinib, and 250 nM to 8 μmol/L vandetanib. Genomic DNA was isolated from resistant clones, and Sanger sequencing was used to identify those that harbored substitutions.
Allografts
Amino Acid Sequence
Anti-Antibodies
Biological Assay
cabozantinib
Cells
Clone Cells
Cloning Vectors
Doxycycline
Epitopes
Genome
Homo sapiens
Immunoblotting
KIF5B protein, human
Lentivirus
Mutagenesis
ponatinib
regorafenib
Transduction, Genetic
vandetanib
Example 4
Given the effect of the overexpression of the SEQ ID NO:1, further experiments were performed to study its effect in cell invasion, another key oncogenic trait. Boyden chamber assay was used to determine invasiveness of A549 and H10 cancer cells after 4 days of doxycycline induction of SEQ ID NO:1, showing that the expression of the micropeptide induces a significant decrease in invasion (
Biological Assay
Carcinogenesis
Cell Lines
Cells
Down-Regulation
Doxycycline
Mesenchyma
N-Cadherins
Neoplastic Cell Transformation
Slugs
Snails
Tumor Suppressor Genes
TWIST1 protein, human
Vimentin
Example 7
In the context of gene therapy, inducible gene expression systems may enable gene expression to be triggered at specific times and in specific cell types. To develop a Tet-on system for inducing Klotho gene expression, the ZFP3_VPR construct was cloned into a doxycycline-inducible vector. The ZFP3_VPR and ZFP52_VPR sequences and the inducible vector (Lenti-iCas9-neo, Addgene #85400) were amplified using Clontech HiFi according to the manufacturers protocol and the following primers:
Cells
Cloning Vectors
DNA-Binding Proteins
Doxycycline
Gene Expression
KL protein, human
Oligonucleotide Primers
Therapy, Gene
pSBtet-BP was a gift from Eric Kowarz (Addgene plasmid # 60496; http://n2t.net/addgene:60496 ; RRID:Addgene_60496) (59 (link)). pCMV(CAT)T7-SB100 was a gift from Zsuzsanna Izsvak (Addgene plasmid # 34879; http://n2t.net/addgene:34879 ; RRID:Addgene_34879) (74 ). An oligonucleotide-encoding eGFP fused to AAK1 were generated by BioBasic Inc, using the sequence of eGFP, followed by the sequence encoding a spacer peptide (GGG GGG TCT GGT GGC AGT GGA GGG GGA TCC), followed by the sequence of human AAK1, as per accession number NM_Q2M2I8. This oligonucleotide sequence was subcloned into pSB-tet-BP to generate pSB-tet-BP-eGFP-AAK1(WT). From this plasmid, mutant AAK1 (S447, T507, S519, T359, T360, Thr448, Thr445, Ser650 to Ala) constructs were derived by site-directed mutagenesis by BioBasic Inc.
pSBtet-BP plasmids encoding various AAK1 WT and AAK1 mutant constructs alongside pCMV(CAT)T7-SB100 were cotransfected into ARPE-19 cells using FuGene HD transfection reagent, as per manufacturer’s protocol (Promega), followed by a selection of stably engineered cells in media supplemented with 2 μg/ml puromycin for a period of 2 to 3 weeks.
pSBtet-BP stable cells were kept in cell culture media as indicated above but were incubated with 10% fetal bovine serum without tetracycline and maintained in 2 μg/ml puromycin. For the induction of AAK1-GFP WT or mutant, doxycycline (Dox) was used at a final concentration of 1 μM in the cell culture media for 24 h before freezing cells for different downstream applications.
pSBtet-BP plasmids encoding various AAK1 WT and AAK1 mutant constructs alongside pCMV(CAT)T7-SB100 were cotransfected into ARPE-19 cells using FuGene HD transfection reagent, as per manufacturer’s protocol (Promega), followed by a selection of stably engineered cells in media supplemented with 2 μg/ml puromycin for a period of 2 to 3 weeks.
pSBtet-BP stable cells were kept in cell culture media as indicated above but were incubated with 10% fetal bovine serum without tetracycline and maintained in 2 μg/ml puromycin. For the induction of AAK1-GFP WT or mutant, doxycycline (Dox) was used at a final concentration of 1 μM in the cell culture media for 24 h before freezing cells for different downstream applications.
Cell Culture Techniques
Cells
Culture Media
Doxycycline
Fetal Bovine Serum
FuGene
Homo sapiens
Mutagenesis, Site-Directed
Oligonucleotides
Peptides
Plasmids
Promega
Puromycin
Somatostatin-Secreting Cells
Tetracycline
Transfection
ARPE-19 cells stably expressing eGFP-CLC were seeded onto glass coverslips the day before the experiment, and in some cases gone through siRNA transfection (Figure 1 , Figure 2 , Figure 3 , Figure 5 , Figure 6 , Figure 7 ). pSBTet-AAK1-GFP WT and pSBTet-AAK1-GFP mutant cells were seeded onto glass coverslips the day before the experiment for siRNA of AAK1 in Figure 3 . After two rounds of siRNA transfection to AAK1, doxycycline was added to the cells during the rest day. Cells were incubated for 4h at 20 μM TMG for or in vehicle (dH20) prior to live-cell microscopy (Fig. 2 ). Cells were either incubated for 3h at 5 μM of LP-935509 or in vehicle control (0.1% (v/v) DMSO) prior to the fixation (Fig. 6 ). Following 1-h serum starvation, cells were treated with 20 ng/ml rhodamine EGF and 10 μg/ml A657-Tfn in combination (Fig. 3 ), followed by immediate fixation in 4% paraformaldehyde. For Figure 7 , cells were seeded onto 6-well plates for 24 prior to incubation in media without glucose, glutamine, and no phenol red (catalog no. A1443001) supplemented with 10% dialyzed fetal bovine serum (Thermo Fisher Scientific), and glucose and/or glutamine, as indicated, for 4 h.
Cells
Doxycycline
Fetal Bovine Serum
Glucose
Glutamine
Microscopy
paraform
Rhodamine
RNA, Small Interfering
Serum
Sulfoxide, Dimethyl
Transfection
Top products related to «Doxycycline»
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Doxycycline is a broad-spectrum antibiotic belonging to the tetracycline class. It inhibits bacterial protein synthesis by binding to the 30S ribosomal subunit. Doxycycline is commonly used in the treatment of various bacterial infections.
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Lipofectamine 2000 is a cationic lipid-based transfection reagent designed for efficient and reliable delivery of nucleic acids, such as plasmid DNA and small interfering RNA (siRNA), into a wide range of eukaryotic cell types. It facilitates the formation of complexes between the nucleic acid and the lipid components, which can then be introduced into cells to enable gene expression or gene silencing studies.
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Fetal Bovine Serum (FBS) is a cell culture supplement derived from the blood of bovine fetuses. FBS provides a source of proteins, growth factors, and other components that support the growth and maintenance of various cell types in in vitro cell culture applications.
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Puromycin is a laboratory product manufactured by Merck Group. It functions as an antibiotic that inhibits protein synthesis in eukaryotic cells.
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DMEM (Dulbecco's Modified Eagle's Medium) is a cell culture medium formulated to support the growth and maintenance of a variety of cell types, including mammalian cells. It provides essential nutrients, amino acids, vitamins, and other components necessary for cell proliferation and survival in an in vitro environment.
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Penicillin/streptomycin is a commonly used antibiotic solution for cell culture applications. It contains a combination of penicillin and streptomycin, which are broad-spectrum antibiotics that inhibit the growth of both Gram-positive and Gram-negative bacteria.
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Doxycycline is an antibiotic medication used to treat a variety of bacterial infections. It is a broad-spectrum tetracycline derivative effective against both Gram-positive and Gram-negative bacteria. Doxycycline functions by inhibiting bacterial protein synthesis, thereby preventing the bacteria from reproducing and causing infection.
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Polybrene is a cationic polymer used as a transfection reagent in cell biology research. It facilitates the introduction of genetic material into cells by enhancing the efficiency of DNA or RNA uptake.
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Doxycycline is a broad-spectrum tetracycline antibiotic used in various laboratory applications. It functions as an inhibitor of bacterial protein synthesis, effectively disrupting the growth and reproduction of targeted microorganisms.
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Doxycycline (DOX) is an antibiotic product manufactured by Merck Group. It is a tetracycline class of antibiotics used for various medical applications. The core function of doxycycline is to inhibit bacterial protein synthesis, thereby preventing the growth and reproduction of bacteria.
More about "Doxycycline"
Doxycycline is a broad-spectrum tetracycline antibiotic that is widely used to treat a variety of bacterial infections.
It is effective against both Gram-positive and Gram-negative bacteria, including Chlamydia, Mycoplasma, Rickettsia, and some protozoa.
Doxycycline is commonly prescribed for respiratory tract infections, skin and soft tissue infections, sexually transmitted diseases, and other conditions.
Doxycycline works by inhibiting bacterial protein synthesis, preventing the bacteria from growing and reproducing.
It is generally well-tolerated, but can cause side effects such as gastrointestinal upset, photosensitivity, and teeth discoloration in children.
Researchers can utilize PubCompare.ai, an AI-driven platform, to optimize doxycycline protocols through comparisons of literature, preprints, and patents.
This helps identify the most reproducible and accurate approaches, enabling researchers to achieve better results.
In addition to doxycycline, researchers may also utilize other common cell culture reagents and techniques, such as Lipofectamine 2000 for transfection, fetal bovine serum (FBS) for cell growth, puromycin for selection, and DMEM or penicillin/streptomycin for cell culture media.
Polybrene may also be used to enhance transduction efficiency.
By leveraging these tools and techniques, along with the power of AI-driven optimization, researchers can streamline their doxycycline research and unlock new insights.
It is effective against both Gram-positive and Gram-negative bacteria, including Chlamydia, Mycoplasma, Rickettsia, and some protozoa.
Doxycycline is commonly prescribed for respiratory tract infections, skin and soft tissue infections, sexually transmitted diseases, and other conditions.
Doxycycline works by inhibiting bacterial protein synthesis, preventing the bacteria from growing and reproducing.
It is generally well-tolerated, but can cause side effects such as gastrointestinal upset, photosensitivity, and teeth discoloration in children.
Researchers can utilize PubCompare.ai, an AI-driven platform, to optimize doxycycline protocols through comparisons of literature, preprints, and patents.
This helps identify the most reproducible and accurate approaches, enabling researchers to achieve better results.
In addition to doxycycline, researchers may also utilize other common cell culture reagents and techniques, such as Lipofectamine 2000 for transfection, fetal bovine serum (FBS) for cell growth, puromycin for selection, and DMEM or penicillin/streptomycin for cell culture media.
Polybrene may also be used to enhance transduction efficiency.
By leveraging these tools and techniques, along with the power of AI-driven optimization, researchers can streamline their doxycycline research and unlock new insights.