To assess direct binding of Eomes to the Pdcd1 promoter, we used a modified version of Cut & Run (Skene et al., 2018 (link)) with a qPCR readout. EL4 cells were nucleofected using an Amaxa cell line Nucleofector kit L (Lonza) with either pCMV-myc T-bet or pCMV-Eomes FLAG plasmids. Following a 48 hr incubation to induce recombinant protein expression, 100,000 EL4 cells were used in the Cut&Run assay. For primary mouse cells, CD8+ splenocytes from d45 Armstrong (TMEM) or d45 clone 13 (TEX) mice were isolated and 10,000 cells were used in the Cut&Run assay. α-myc (Invitrogen) or α-FLAG (Invitrogen) antibodies were used to IP myc-T-bet or Eomes-FLAG proteins in EL4 cells. α-T-bet (4B10, eBioscience) or α-Eomes (Dan11mag, eBioscience) was used to IP T-bet or Eomes from primary mouse splenocytes. Immunoprecipitated DNA was subjected to two rounds of nested PCR using the following oligo pairs: I–forward 5′-actctaacatgccacaaaaccatag, reverse 5′-cttccagttttatacctgatcgaag (Cruz-Guilloty et al., 2009 (link)), Pdcd1–5’-ccttgctcctcaccacactgc, reverse 5′-cagagcagatcatgaggactg (Kao et al., 2011 (link)), and Il4–forward 5′-gagttaaagttgctgaaaccaagg, reverse 5′-attttccaattggtctgatttcac (Cruz-Guilloty et al., 2009 (link)).
α flag
Manufactured by Thermo Fisher Scientific
The α-FLAG is a lab equipment product designed for protein purification and detection. It functions as an affinity tag that can be fused to a target protein, allowing for efficient isolation and identification of the protein of interest.
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Lab products found in correlation
α myc, by Thermo Fisher Scientific (2 mentions)
Amaxa cell line nucleofector kit l, by Lonza (2 mentions)
α eomes dan11mag, by Thermo Fisher Scientific (2 mentions)
α t bet 4b10, by Thermo Fisher Scientific (2 mentions)
Anti mycn antibody, by Santa Cruz Biotechnology (2 mentions)
Jetprime, by Polyplus Transfection (2 mentions)
β actin, by Merck Group (2 mentions)
V5 ip, by Thermo Fisher Scientific (2 mentions)
Kelly cells, by Santa Cruz Biotechnology (2 mentions)
Anti eya1 antibody, by ProSci (2 mentions)
Kelly cells, by Merck Group (2 mentions)
α rps6, by Abcam (1 mentions)
α phosphos6, by Cell Signaling Technology (1 mentions)
α g6pdh, by Merck Group (1 mentions)
Vectashield mounting media, by Vector Laboratories (1 mentions)
6 protocols using α flag
1
Cut&Run Assay for Eomes-Pdcd1 Binding
2
Cut&Run Assay for Eomes-Pdcd1 Binding
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To assess direct binding of Eomes to the Pdcd1 promoter, we used a modified version of Cut & Run (Skene et al., 2018 (link)) with a qPCR readout. EL4 cells were nucleofected using an Amaxa cell line Nucleofector kit L (Lonza) with either pCMV-myc T-bet or pCMV-Eomes FLAG plasmids. Following a 48 hr incubation to induce recombinant protein expression, 100,000 EL4 cells were used in the Cut&Run assay. For primary mouse cells, CD8+ splenocytes from d45 Armstrong (TMEM) or d45 clone 13 (TEX) mice were isolated and 10,000 cells were used in the Cut&Run assay. α-myc (Invitrogen) or α-FLAG (Invitrogen) antibodies were used to IP myc-T-bet or Eomes-FLAG proteins in EL4 cells. α-T-bet (4B10, eBioscience) or α-Eomes (Dan11mag, eBioscience) was used to IP T-bet or Eomes from primary mouse splenocytes. Immunoprecipitated DNA was subjected to two rounds of nested PCR using the following oligo pairs: I–forward 5′-actctaacatgccacaaaaccatag, reverse 5′-cttccagttttatacctgatcgaag (Cruz-Guilloty et al., 2009 (link)), Pdcd1–5’-ccttgctcctcaccacactgc, reverse 5′-cagagcagatcatgaggactg (Kao et al., 2011 (link)), and Il4–forward 5′-gagttaaagttgctgaaaccaagg, reverse 5′-attttccaattggtctgatttcac (Cruz-Guilloty et al., 2009 (link)).
3
Fluorescent Detection of Protein Interactions in E. coli
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E. coli strain C43(DE3)62 (link) cells were co-transformed with pET26 N-terminal His-tagged HsmA and pET52 C-terminal FLAG-tagged HphA or empty pET26 and pET52 C-terminal FLAG-tagged HphA. Cells were grown overnight at 37 °C in auto-induction media63 (link) supplemented with ampicillin and kanamycin. Cells were harvested by centrifugation at 1500 × g, and washed with PBS containing 0.9 mM MgCl2. Cells were incubated with α-FLAG (1:200 dilution from rabbit serum; Thermo) for 1 h at room temperature, followed by washing in PBS + 0.9 mM MgCl2. Cells were incubated with R-PE conjugated anti-rabbit IgG (Rockland) diluted 1:200 in PBS + 0.9 mM MgCl2 for 1 h at room temperature, followed by washing with PBS + 0.9 mM MgCl2 to remove excess antibodies. Cells were resuspended in 200 µL PBS + 0.9 mM MgCl2, and fluorescence measurements recorded using a BioTek Synergy plate reader with a 488 nm excitation and 575 nm emission.
4
Coimmunoprecipitation of MYCN and EYA1 in KELLY cells
5
Protein Quantification and Localization Assay
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The antibodies used are as follows: α-RPS6 (Abcam), α-phosphoS6 (Cell Signaling), α-FLAG (Stratagene, La Jolla, CA), goat α-rabbit HRP-conjugated secondary (The Jackson Laboratory), α-HA and α-Myc A14 (Santa Cruz Biotechnology), α-G6PDH (Sigma, St. Louis, MI), α-FLAG (ThermoFisher), and goat α-rabbit FITC-conjugated secondary (Rockland). All histone antibodies were purchased from Active Motif. For confocal microscopy, Vectashield mounting media containing DAPI were purchased from Vector Laboratories (Burlingame, CA).
6
Coimmunoprecipitation of MYCN and EYA1 in KELLY cells
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KELLY cells were purchased from Sigma (St. Louis, MO) and cultured in DMEM:F12, supplemented with 10% FBS. HEK293TN cells were cultured in DMEM with 10% FBS. Transient transfection was performed by using jetPRIME (Polyplus transfection, #114-07) per the manufacturer’s instructions. CoIP analysis was performed essentially as previously described[21 (link)]. Briefly, two days post-transfection, whole cell lysates of HEK293TN cells were prepared and used for V5-IP (Invitrogen, #R960-25). In the case of KELLY cells, nuclear extracts were isolated and subjected to immunoprecipitation with anti-MYCN antibody (Santa Cruz, #sc-53993), followed by immunoblotting with anti-EYA1 antibody (Prosci, #25-067). The following antibodies were used in western blot: α-Flag (Thermo Fisher Scientific, #MA1-91878) and β-actin (Sigma, #A5316).
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