The following reagents and antibodies were purchased from commercial sources: inhibitor cocktail (Trichostatin A (TSA, T8552, Sigma), protease inhibitor cocktail (P8340, Sigma), phosphatase inhibitor cocktail (P0044, Sigma)), fedratinib (S2736, Selleckchem), universal nuclease (88700, Thermo Fisher), Bradford assay (23200, Thermo Fisher), dithiothreitol (DTT; DTT100, Goldbio), enzyme-linked chemiluminescence (ECL) plus (32132, Thermo Fisher), SYBR Green PCR Master Mix (4472908, Applied Biosystems), streptavidin agarose (20359, Thermo Fisher), Protein A/G PLUS-Agarose (sc-2003, Santa Cruz Biotechnology), anti-Flag agarose gel (A2220, Sigma) and anti-HA affinity gel (E6779, Sigma). Antibodies were as follows: STAT3 (9139, CST), phospho-STAT3 (Tyr705) (ab76315, Abcam), β-actin (C4) HRP (SC-47778, Santa Cruz), Na/K-ATPase (SC-21712, Santa Cruz), histone H3 (4499S, CST), Flag HRP (A8592, Millipore), HA-probe (Y-11) (SC805, Santa Cruz), HA-probe (F-7) (SC7392, Santa Cruz), DHHC7 (ab138210, Abcam), DHHC7 (R12–3691, Assay Biotechnology), Alexa Fluor 350 goat anti-rabbit IgG (A-11046, Invitrogen), Alexa Fluor 594 goat anti-mouse IgG (8890S, CST), mouse CD4 PerCP-Cy5.5 (560767, BD Pharmingen), mouse IL-17A PE (560767, BD Pharmingen), anti-mouse IgG HRP (7076S, CST) and anti-rabbit IgG HRP (7074S, CST).
Anti flag agarose gel
Manufactured by Merck Group
Anti-Flag agarose gels are a type of lab equipment used for the purification and isolation of FLAG-tagged proteins. They provide a reliable and efficient method for the affinity-based separation of proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Ecl plus, by Thermo Fisher Scientific (3 mentions)
Enhanced chemiluminescence, by Merck Group (2 mentions)
Protease inhibitor cocktail, by Roche (2 mentions)
Dynabeads protein g, by Thermo Fisher Scientific (2 mentions)
Sds loading buffer, by Solarbio (2 mentions)
Streptavidin agarose, by Thermo Fisher Scientific (1 mentions)
Anti ha affinity gel, by Merck Group (1 mentions)
Universal nuclease, by Thermo Fisher Scientific (1 mentions)
Phosphatase inhibitor cocktail, by Merck Group (1 mentions)
Na k atpase, by Santa Cruz Biotechnology (1 mentions)
Bradford assay, by Thermo Fisher Scientific (1 mentions)
Ha probe f 7, by Santa Cruz Biotechnology (1 mentions)
Fedratinib, by Selleck Chemicals (1 mentions)
β actin c4 hrp, by Santa Cruz Biotechnology (1 mentions)
Ha probe y 11, by Santa Cruz Biotechnology (1 mentions)
Phospho stat3 tyr705, by Abcam (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
Sybr green pcr master mix, by Thermo Fisher Scientific (1 mentions)
Protease inhibitor cocktail, by Merck Group (1 mentions)
Anti ha, by Thermo Fisher Scientific (1 mentions)
9 protocols using anti flag agarose gel
1
Immunoprecipitation and Western Blot Analysis
2
Cell Lysis and Protein Detection
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Cell pellets were lysed in RIPA buffer (50 mM Tris, pH7.4, with 150 mM NaCl, 1% NP40, 9 mM ethylenediamine tetra acetic acid (EDTA)) and boiled for 30 min in a 100°C water bath. The following antibodies were used to detect protein expression: anti-HA from Invitrogen (Carlsbad, CA), anti-Flag agarose gel from Sigma, anti-V5 and anti-EGFP from Invitrogen, and anti-tubulin from Abcam (Cambridge, MA). All antibodies were used according to the manufacturers’ protocols.
3
Immunoprecipitation and Western Blotting
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For immunoprecipitation, whole-cell lysis was carried out by incubating the cells overnight with the corresponding antibodies and Protein A/G beads (Pierce). For immunoprecipitation with anti-FLAG, whole-cell lysis was performed by incubating the cells overnight with anti-Flag agarose gel (Sigma). The beads were then washed six times with low salt lysis buffer. The isolated protein was then eluted with 1 × SDS loading buffer, separated by SDS-PAGE (10%), and transferred to a PVDF membrane. Then, incubated with the appropriate antibodies (TBK1, HNF1A, HA, and Flag) and membranes were detected using enhanced chemiluminescence (ECL) detection system. The antibodies used in this study are listed in Table S5 .
4
Immunoprecipitation and Western Blotting Protocol
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Cells were extracted in an ice-cold low-salt lysis buffer [50 mM Hepes (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100] supplemented with a protease inhibitor cocktail (5 mg/ml; Roche). A 20-μl aliquot of each sample was subjected to SDS–polyacrylamide gel electrophoresis. For immunoprecipitation experiments, whole-cell extracts were incubated with anti-Flag agarose gels (Sigma) overnight. The beads were washed three times with low-salt lysis buffer. As for the re-coimmunoprecipitation experiments, samples after one-time immunoprecipitation were boiled for 5 min with 10% SDS. The supernatant of samples was then diluted 10 times and used for second-time immunoprecipitation with anti-Flag beads. The immunoprecipitates were resuspended in 3× SDS loading buffer (FD Biotechnology) and boiled for 5 min. The released proteins were electrophoresed on 8 to 12% SDS-polyacrylamide gels and transferred onto polyvinylidene difluoride membranes, with subsequent blocking using 5% skim milk. The membranes were incubated with the indicated antibodies and detected using enhanced chemiluminescence (Millipore).
5
Immunoprecipitation and Western Blot Analysis
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For immunoprecipitation experiments, whole-cell lysates were prepared as the method of immunoblot assay, followed by incubation with the anti-Flag agarose gels (Sigma) overnight at 4 °C. The beads were washed five times with low-salt lysis buffer, and then resuspended with 2 × SDS Loading Buffer (Solarbio), and boiled for 10 min. The released proteins were subjected to western blot analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were incubated with indicated antibodies overnight at 4 °C, and added with 20 μl Dynabeads protein G (Invitrogen) for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were incubated with indicated antibodies overnight at 4 °C, and added with 20 μl Dynabeads protein G (Invitrogen) for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
6
Protein-Protein Interaction Detection
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To detect the protein–protein interaction, the immunoprecipitation experiments were performed. Whole cell lysates were prepared as the method of immunoblot assay, and incubated with the anti-Flag agarose gels (Sigma) on roller shaker overnight at 4 °C. The beads were washed six times with low salt lysis buffer, re-suspended with 2× SDS Loading Buffer (Solarbio), and boiled for 10 min. The released proteins were subjected to western blot analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were obtained as the method of immunoblot assay, and then incubated with indicated antibodies overnight at 4 °C. Twenty microliter Dynabeads protein G (Invitrogen) were added to the extracted cell proteins and incubated for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
For endogenous immunoprecipitation experiments, the extracted cell proteins were obtained as the method of immunoblot assay, and then incubated with indicated antibodies overnight at 4 °C. Twenty microliter Dynabeads protein G (Invitrogen) were added to the extracted cell proteins and incubated for another 2 h. Then the beads were washed and subjected to western analyses with the indicated antibodies.
7
Cell Lysis and Immunoprecipitation Protocol
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Cells were extracted in ice-cold low-salt lysis buffer (50 mM Hepes pH 7.5, 150 mM NaCl, 1 mM EDTA, 1.5 mM MgCl2, 10% glycerol, 1% Triton X-100) supplemented with 5 mg/mL protease inhibitor cocktail (Roche). A 20-μL aliquot of each sample was subjected to SDS-PAGE. For immunoprecipitation (IP) experiments, whole-cell extracts were incubated with anti-Flag agarose gels (Sigma) overnight. The beads were washed three times with low-salt lysis buffer. The immunoprecipitates were resuspended in 3×SDS Loading Buffer (FD Biotechnology) and boiled for 5 minutes. The released proteins were electrophoresed on 8–12% SDS-polyacrylamide gels and transferred onto PVDF membranes, with subsequent blocking using 5% skim milk. The membranes were incubated with the indicated antibodies and detected using enhanced chemiluminescence (Millipore).
8
Immunoprecipitation Assay Protocol
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For immunoprecipitation, whole-cell extracts were prepared after infection, transfection, or stimulation with indicated ligands, followed by incubation overnight with anti-Flag agarose gels (Sigma) or appropriate antibody with protein A/G agarose gels (Pierce) at 4°C. Beads were subsequently washed 3–5 times using low-salt lysis buffer (50 mM HEPES, 150 mM NaCl, 1 mM EDTA, 10% glycerol, 1.5 mM MgCl2, and 1% Triton X-100), and immunoprecipitates were eluted with × 2 SDS loading buffer and resolved by SDS-PAGE. Then proteins were transferred to PVDF membranes (Bio-Rad) and further incubated with the appropriate antibodies. Immobilon Western Chemiluminescent HRP Substrate (Millipore, WBKLS0500) was used for protein detection.
9
Endogenous and Exogenous Protein Interactions
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For endogenous interaction, BMDM cells in each experimental group were lysed in NP-40 buffer mixed with protease inhibitors and centrifuged at 4°C for 15 min. The cell lysates were collected and incubated with IP antibodies [NEK7 (1:100) or ASC (1:100)] overnight at 4°C. Protein A/G beads were added and incubated for 4 h at 4°C. Protein A/G beads pulled down the target protein for detection using Western blotting. For exogenous interaction, HEK-293T cells were transfected with Lipofectamine 2000 according to the kit instructions. After 24 h, cells were collected and lysed in NP-40 buffer mixed with protease inhibitors. Cell lysates were immunoprecipitated with the anti-Flag agarose gels (1:100, Sigma, A2220) and detected using Western blotting.
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