Cells were cultured on coverslips, fixed with 4% buffered paraformaldehyde, and then permeabilized with 0.1% Triton X-100 in phosphate-buffered saline (PBS). Cells were blocked with Dako Protein Block (Dako) for 1 h and incubated with antibody to Flag (Sigma) diluted in Dako Protein Block. Alexa 488-labeled donkey anti-mouse was used as a secondary antibody. Cells were counterstained with DAPI, rinsed with PBS, mounted with Gelvatol, and examined using a ZEISS confocal microscope LSM700.
Antibody to flag
Manufactured by Merck Group
Sourced in China
Antibody to Flag is a laboratory reagent used to detect and purify proteins that have been engineered to contain a specific peptide sequence known as a 'flag' tag. The antibody specifically binds to this tag, allowing researchers to isolate and identify the target protein.
Automatically generated - may contain errors
Lab products found in correlation
Sorafenib, by Selleck Chemicals (2 mentions)
Vertepor n, by Selleck Chemicals (2 mentions)
Antibody to gapdh, by Santa Cruz Biotechnology (2 mentions)
Histone h3, by Cell Signaling Technology (2 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (2 mentions)
Bcl xl, by Cell Signaling Technology (2 mentions)
Hepg2, by American Type Culture Collection (2 mentions)
Dako protein block, by Agilent Technologies (1 mentions)
Dna dye dapi, by Vector Laboratories (1 mentions)
Fugene 6 transfection reagent, by Promega (1 mentions)
Neuro 2a cells, by American Type Culture Collection (1 mentions)
Ab76307, by Abcam (1 mentions)
Gammabind g sepharose beads, by GE Healthcare (1 mentions)
Aceq universal sybr qpcr master mix, by Vazyme (1 mentions)
Gapdh, by Proteintech (1 mentions)
Phospho raptor s792, by Cell Signaling Technology (1 mentions)
Phospho p70 s6 kinase, by Cell Signaling Technology (1 mentions)
Phospho s6 ribosomal protein, by Cell Signaling Technology (1 mentions)
Antibody to β actin, by Merck Group (1 mentions)
Antibody to γ tubulin, by Abcam (1 mentions)
8 protocols using antibody to flag
1
Immunofluorescence Microscopy of Flag-tagged Proteins
2
Evaluating TRPC3 Effects on Neuro-2a Cells
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Neuro-2a
cells (American Type Culture Collection) were grown under standard
conditions. Transfections were performed using Fugene 6 Transfection
Reagent (Promega) according to the manufacturer’s instructions.
For cell death assays, cells transfected with FLAG-TRPC3 constructs
were fixed after 24 h with 4% paraformaldehyde and subjected to indirect
immunofluorescence using an antibody to FLAG (1:500, Sigma). Cell
survival and death were assessed on the basis of cell integrity and
the morphology of the nucleus as determined using the DNA dye DAPI
(Vector Laboratories). Cell counts were conducted in a blinded manner,
and 50–100 cells were counted per condition. Cells were acquired
from three independent experiments. For NFAT translocation assays,
cells transfected with FLAG-TRPC3 constructs alongside GFP-tagged
NFAT were fixed after 18 h with 4% paraformaldehyde and subjected
to indirect immunofluorescence using antibodies to FLAG (1:500, Sigma),
GFP (1:500, Clontech), and the DNA dye DAPI (Vector Laboratories).
Cytoplasmic and nuclear localization of GFP-NFAT were scored in 50–100
FLAG-positive cells. Cells were acquired from three independent experiments.
Data were analyzed using GraphPad Prism.
cells (American Type Culture Collection) were grown under standard
conditions. Transfections were performed using Fugene 6 Transfection
Reagent (Promega) according to the manufacturer’s instructions.
For cell death assays, cells transfected with FLAG-TRPC3 constructs
were fixed after 24 h with 4% paraformaldehyde and subjected to indirect
immunofluorescence using an antibody to FLAG (1:500, Sigma). Cell
survival and death were assessed on the basis of cell integrity and
the morphology of the nucleus as determined using the DNA dye DAPI
(Vector Laboratories). Cell counts were conducted in a blinded manner,
and 50–100 cells were counted per condition. Cells were acquired
from three independent experiments. For NFAT translocation assays,
cells transfected with FLAG-TRPC3 constructs alongside GFP-tagged
NFAT were fixed after 18 h with 4% paraformaldehyde and subjected
to indirect immunofluorescence using antibodies to FLAG (1:500, Sigma),
GFP (1:500, Clontech), and the DNA dye DAPI (Vector Laboratories).
Cytoplasmic and nuclear localization of GFP-NFAT were scored in 50–100
FLAG-positive cells. Cells were acquired from three independent experiments.
Data were analyzed using GraphPad Prism.
3
ChIP Assay for Histone Modifications
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatin immunoprecipitation (ChIP) assays were performed as previously described (41 (link)). Briefly, tissues were fixed with 1% formaldehyde for 15 min, and then treated with 125 mM glycine for 5 min to stop the cross-linking reaction. After harvest, cross-linked tissues were grinded (0.5 g) and re-suspended in 6 ml lysis buffer containing protease inhibitors. Chromatin was sheared by sonication to ∼500 bp fragments. 1 ml (2 mg/ml) protein was used as per immunoprecipitation and 10 μl was kept as the input DNA. ChIP was performed with 6 μl antibody to FLAG (Sigma), 10 μl antibody to HP1, 10 μl antibody to H2A.Z, 3 μl antibody to acetylated H3K56 (ab76307; Abcam) or 3 μl antibody to H3 (2650; CST). Immunoprecipitated DNA was enriched with the GammaBind G Sepharose beads (17-0885-02; GE Healthcare) and eluted using elution buffer. Finally, purified DNA was quantified by real-time qPCR (7500; ABI) with AceQ® Universal SYBR qPCR Master Mix (Q511; Vazyme) and primer pairs (see Supplementary Table S2 ). Occupancies were normalized by the input DNA and presented as a percentage of input DNA. H3K56 acetylation data need to be further normalized to histone H3 level.
4
Western Blot Antibody Panel Analysis
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Western blot was performed as described previously [39 (link)]. Antibodies to PPARγ (C26H12) (2435S) and HA (C29F4) (3724S) were purchased from Cell Signaling Technology (Danvers, Massachusetts, USA); antibodies to SIAH2, CD36 and GAPDH were purchased from Proteintech (Wuhan, China); antibody to FABP2 was purchased from ABclonal (Wuhan, China); antibody to Lamin B1 was purchased from Abways Technology (Shanghai, China); and antibody to FLAG was purchased from Sigma-Aldrich (Saint Louis, MO, USA).
5
Antibody Validation for Cell Signaling
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antibodies used in this study included antibody to Flag (Sigma), antibody to Myc (Clone 9E10 Santa Cruz Biotechnology), mouse antibody to GFP (B-2, Santa Cruz Biotechnology), rabbit antibody to GFP (MBL), antibody to RAPTOR (Cell Signaling), rabbit antibody to VHL (Santa Cruz Biotechnology FL-181 and Cell Signaling), antibody to β-actin (Sigma), antibody to γ-tubulin (Sigma), rabbit antibody to V5 (Millipore), and mouse antibody to HA Clone12CA5 (Roche). The antibodies to mTOR, p70 S6 kinase, phospho-p70 S6 Kinase, phospho-S6 ribosomal protein, phospho-RAPTOR S792, REDD1 and phospho-drosophila p70 S6 (Thr398) kinase were all obtained from Cell Signaling, the antibody against HIF1α from BD Transduction Laboratories and the antibody against HIF2α from Abcam.
6
Cell Culture and Reagent Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture and reagents Huh-7, HepG2 and LO2 cells were obtained from the American Type Culture Collection. These cell lines were maintained in Dulbecco's Modi ed Eagle Medium (DMEM; GIBCO BRL) supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 U/mL streptomycin. Cultures were maintained at 37℃ in a humidi ed at mosphere with 5% CO2. Sorafenib and Vertepor n were purchased from Selleck Chemicals.
Antibody to PARP, YAP, p-YAP(S127), survivin, Bcl-xl and Histone H3 were purchased from Cell Signaling Technology, Inc.; antibody to GAPDH was from Santa Cruz Biotechnology, Inc.; antibody to Flag was from Sigma.
Antibody to PARP, YAP, p-YAP(S127), survivin, Bcl-xl and Histone H3 were purchased from Cell Signaling Technology, Inc.; antibody to GAPDH was from Santa Cruz Biotechnology, Inc.; antibody to Flag was from Sigma.
7
Cell Culture and Reagent Protocols
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell culture and reagents Huh-7, HepG2 and LO2 cells were obtained from the American Type Culture Collection. These cell lines were maintained in Dulbecco's Modi ed Eagle Medium (DMEM; GIBCO BRL) supplemented with 10% (v/v) FBS, 100 U/mL penicillin and 100 U/mL streptomycin. Cultures were maintained at 37℃ in a humidi ed at mosphere with 5% CO2. Sorafenib and Vertepor n were purchased from Selleck Chemicals.
Antibody to PARP, YAP, p-YAP(S127), survivin, Bcl-xl and Histone H3 were purchased from Cell Signaling Technology, Inc.; antibody to GAPDH was from Santa Cruz Biotechnology, Inc.; antibody to Flag was from Sigma.
Antibody to PARP, YAP, p-YAP(S127), survivin, Bcl-xl and Histone H3 were purchased from Cell Signaling Technology, Inc.; antibody to GAPDH was from Santa Cruz Biotechnology, Inc.; antibody to Flag was from Sigma.
8
Cell Culture and Plasmid Modification Protocol
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
Revolutionizing how scientists
search and build protocols!