PANC-1 cells were transfected with Id3-Flag plasmids using Reagent (Invitrogen). After 6 hours of incubation at 37°C, growth media was changed. Tamoxifen was added to concentration of 4uM after 24 hours. After incubation with tamoxifen for 48 hours, cells were harvested using cell lysis buffer [20mM TrisHCl (pH7.5, 1% Triton X-100, 10% glycerol, 300mM NaCl, 0.5mM EDTA (pH8.0), 1mM Na3VO4, protease inhibitors) for immunoprecipitation. The FLAG fusion proteins were extracted using anti-FLAG M2 Magnetic Beads, proteins were electrophorectically separated on SDS-4–12% polyacrylamide gels (Thermo Scientific), transferred onto nitrocellulose membranes (LI-COR Biosciences) blocked with Odyssey Blocking Buffer (LI-COR Biosciences) incubated at RT with anti-FLAG (Abcam) and anti-beta-actin (Abcam) as control. Secondary antibody detection was performed with IRDye conjugates (LI-COR Biosciences).
Anti flag m2 magnetic beads
Manufactured by Thermo Fisher Scientific
Anti-FLAG M2 Magnetic Beads are agarose beads conjugated with an anti-FLAG M2 monoclonal antibody. They are used for the purification and detection of FLAG-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Sds 4 12 polyacrylamide gels, by Thermo Fisher Scientific (2 mentions)
Odyssey blocking buffer, by LI COR (2 mentions)
Nitrocellulose membrane, by LI COR (2 mentions)
Anti β actin, by Abcam (2 mentions)
Anti flag, by Abcam (2 mentions)
Irdye conjugates, by LI COR (1 mentions)
Anti c myc magnetic beads, by Thermo Fisher Scientific (1 mentions)
Anti flag m2 magnetic bead, by Merck Group (1 mentions)
Proteina magnetic beads, by Abcam (1 mentions)
Anti ha magnetic beads, by Thermo Fisher Scientific (1 mentions)
Protein a magnetic beads, by Thermo Fisher Scientific (1 mentions)
Ripa buffer, by Abcam (1 mentions)
Protease inhibitor cocktail, by Abcam (1 mentions)
Anti actin, by Abcam (1 mentions)
Anti hmgcr, by Abcam (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Fetal calf serum (fcs), by Thermo Fisher Scientific (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
11 protocols using anti flag m2 magnetic beads
1
Immunoprecipitation of Id3-Flag Fusion Proteins
2
Endogenous and Exogenous Co-Immunoprecipitation of RPLP1 and NS4B
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For immunoprecipitation of endogenous RPLP1, PK15 cells transfected with the plasmids pcDNA3.1-NS4B-Flag were lysed by WB and IP lysis buffer (containing 1 mM PMSF) on ice for 0.5 h. Lysates were collected into microcentrifuge tube and centrifugated at 4°C for 0.5 h. The anti-Flag-M2 magnetic beads (#M8823, Sigma Aldrich) were placed in magnetic separator to discard the storage buffer. After washed with TBS, the equilibrated magnetic beads were co-incubated with the supernatant of protein extracts at RT for 2 h with gentle mixing to capture the Flag fusion proteins. Finally, the magnetic beads were collected with magnetic separator and washed five times with TBS followed by detecting target protein with WB.
For exogenous co-IP, HEK-293 T cells co-transfected with the plasmids pcDNA3.1-NS4B-Flag and pcDNA3.1-RPLP1-Myc. Simultaneously, cells co-transfected with pcDNA3.1-NS4B-Flag and pcDNA3.1-Myc as well as pcDNA3.1-RPLP1-Myc and pcDNA3.1-Flag were set as control. At 48 h post transfection (hpt), the cells were lysed and the supernatant was co-incubated with equilibrated anti-Flag-M2 magnetic beads or anti-c-Myc magnetic beads (#88842, Thermo Fisher Scientific), and the subsequent steps were identical to those of endogenous co-IP assay.
For exogenous co-IP, HEK-293 T cells co-transfected with the plasmids pcDNA3.1-NS4B-Flag and pcDNA3.1-RPLP1-Myc. Simultaneously, cells co-transfected with pcDNA3.1-NS4B-Flag and pcDNA3.1-Myc as well as pcDNA3.1-RPLP1-Myc and pcDNA3.1-Flag were set as control. At 48 h post transfection (hpt), the cells were lysed and the supernatant was co-incubated with equilibrated anti-Flag-M2 magnetic beads or anti-c-Myc magnetic beads (#88842, Thermo Fisher Scientific), and the subsequent steps were identical to those of endogenous co-IP assay.
3
Id3-Flag Protein Immunoprecipitation and Detection
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The PANC-1 cells were transfected with Id3-Flag plasmids using Reagent (Invitrogen). After 6 hours of incubation at 37 °C, growth media were changed. Tamoxifen was added to concentration of 4 μm. After incubation with tamoxifen for 48 hours, cells were harvested using cell lysis buffer (TrisHCl, 20 mM [pH, 7.5]; Triton X-100, 1%; glycerol, 10%; NaCl, 300 mM; EDTA, 0.5 mM [pH8.0]; Na3VO4, 1 mM; protease inhibitors] for immunoprecipitation. The FLAG fusion proteins were extracted using anti-FLAG M2 Magnetic Beads, proteins were electrophoretically separated on SDS-4–12% polyacrylamide gels (Thermo Scientific), transferred onto nitrocellulose membranes (LI-COR Biosciences), blocked with Odyssey Blocking Buffer (LI-COR Biosciences), and incubated at RT with anti-FLAG (Abcam) and anti-β-actin (Abcam) as control. Secondary antibody detection was performed with infrared dye conjugates (LI-COR Biosciences).
4
Characterization of p65 Binding to DNA
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EMSA was conducted as previously described.56 (link) Three pairs of 6-carboxy-fluorescein (FAM)-labeled double-stranded DNA probes containing a putative p65-binding site (human, mouse and rat) were generated by annealing their respective complementary oligonucleotides. Probe sequences were shown in Table S5 .
HEK293T cells were transfected with pcDNA3.1-p65-Flag or pcDNA3.1-GATA4-Flag plasmids. After 36 h of transfection, cells were harvested and lysed in 0.5% NP-40 buffer, and Flag-tagged proteins were enriched using anti-FLAG M2 magnetic beads (Thermo Fisher Scientific). The FAM-labeled probe (1 pmol) and 20 μg of immunoprecipitated protein were incubated in reaction buffer containing 5 mM MgCl2, 2 mM EDTA, 50 ng/μL poly (dI-dC), 2.5% glycerol, and 0.5 mg/mL BSA for 20 min at 25°C. The reaction mixture without the immunoprecipitated protein was served as a negative control. For the cold competition assay, 50 pmol and 100 pmol unlabelled probes were added into the reaction. The samples were subjected to 10% non-denaturing PAGE and analyzed using a Typhoon FLA 9500 scanner.
HEK293T cells were transfected with pcDNA3.1-p65-Flag or pcDNA3.1-GATA4-Flag plasmids. After 36 h of transfection, cells were harvested and lysed in 0.5% NP-40 buffer, and Flag-tagged proteins were enriched using anti-FLAG M2 magnetic beads (Thermo Fisher Scientific). The FAM-labeled probe (1 pmol) and 20 μg of immunoprecipitated protein were incubated in reaction buffer containing 5 mM MgCl2, 2 mM EDTA, 50 ng/μL poly (dI-dC), 2.5% glycerol, and 0.5 mg/mL BSA for 20 min at 25°C. The reaction mixture without the immunoprecipitated protein was served as a negative control. For the cold competition assay, 50 pmol and 100 pmol unlabelled probes were added into the reaction. The samples were subjected to 10% non-denaturing PAGE and analyzed using a Typhoon FLA 9500 scanner.
5
Immunoprecipitation of FLAG- and Myc-tagged Proteins
6
NLRP3 Polyubiquitination Assay
7
Immunoprecipitation and Co-immunoprecipitation Protocols
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For immunoprecipitation, cells were lysed with RIPA buffer (Abcam) containing 1X protease inhibitor cocktail (SIGMA) on ice for 30 min. The supernatant was diluted using co-IP lysis buffer, and incubated with primary antibodies over night at 4 C. For co-immunoprecipitation, cells were lysed with co-IP lysis buffer containing 1X protease inhibitor cocktail on ice for 1 h. The supernatant was directly incubated with primary antibodies over night at 4 C. The second day, samples were mixed with Protein A Magnetic Beads (SIGMA), and incubated for 2 h at 4 C. After washed with washing buffer for 3 times, the beads were re-suspended with 30 mL RIPA buffer (Abcam) and 10 mL 4X loading buffer, and denatured for 5 min at 100 C, followed by immunoblotting assays. For Flag-, HA-tag IP or co-IP, anti-FLAG M2 Magnetic Beads (SIGMA) or anti-HA Magnetic Beads (Thermo Fisher Scientific) were used, instead of primary antibodies and Protein A Magnetic Beads (SIGMA).
8
Arsenite Regulates HMGCR Ubiquitination
9
Flag-Tagged MED25 Mutant Expression
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The human pCDNA3-Flag-NLS-MED25-Y39C mutant was generated by PCR using the previously described human pCDNA3-Flag-NLS-MED25 as a template (Verger et al. 2013 (link)) (primer sequences and detailed procedures are available upon request). 293-HEK cells were cultured in DMEM supplemented with 10 % FCS (Gibco BRL). 6 × 10 6 cells were plated in 100 mm tissue culture dishes and transfected with 1 µg of MED25 expression vectors using the Lipofectamine 2000 procedure (Lifetechnologies) for 18 h. Transfected cells were lysed in lysis buffer (50 mM Tris/ HCl, pH 8, 250 mM NaCl, 1 mM EDTA, 1 mM TCEP, 10 % glycerol, 3 mM MgCl 2 , 0.1 % Igepal CA-630). After centrifugation, extracted proteins were immunoprecipitated overnight with anti-FLAG M2 magnetic beads (Thermo) at 4 °C. After extensive washing, the fished proteins were detected by Western blot. The antibodies used were from Sigma (anti-Flag M2), from Santa-cruz (anti-Med6 and anti-Cdk8) or from Bethyl laboratories (anti-Med1, anti-Med14, anti-Med16, anti-Med23 and anti-Med24).
10
Immunoprecipitation of FLAG-tagged Proteins
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