The following antibodies were from Cell Signaling Technology: anti-Casp8 (9746), anti-Cap3 (9664), anti-cIAP2 (3136), anti-pSAPK/JNK (JNK1/2, 4668), anti-pIκBα (9246), anti-Caveolin1 (3267), and anti-cFLIP (56343). The following antibodies were from Abcam: anti-pMLKL (187091), anti-RIPK3 (72106), anti-pRIPK3 (209384), and anti-FADD (108601). Anti-Ub (S.C8017), and protein A/G-conjugated beads were from Santa Cruz Biotechnology, anti-total MLKL (M6697) was from Sigma-Aldrich, anti-RIPK1 (610459) was from BD Medical Technology, and anti-cIAP1 was a gift from of John Silke (Walter and Eliza Hall Institute of Medical Research). z-VAD-fmk was from Enzo Life Sciences, RIPK1 inhibitor GSK’963, RIPK3 inhibitor GSK’840 and IAP antagonist SMAC007, as well as the pRIP1 S166-specific antibody, were provided by GlaxoSmithKline34 (link). IAP antagonist BV6 was provided by Domogoj Vucic (Genentech), recombinant human TNF was from R&D or from PeproTech, Flag-Tagged TNF was from Enzo, necrosulfonamide was from CalBiochem, TLCK (Tosyl-L-lysyl-chloromethane hydrochloride) was from Abcam, cycloheximide (CH) was from Sigma-Aldrich, and Carfilzomib was from BioVision.
Flag tagged tnf
Manufactured by Enzo Life Sciences
Flag-Tagged TNF is a recombinant protein product that contains the tumor necrosis factor (TNF) sequence with a Flag tag added. The Flag tag is a peptide sequence that can be used for the detection and purification of the protein. This product provides a tool for researchers studying the TNF protein and its interactions.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human tnf, by R&D Systems (1 mentions)
Anti p mlkl, by Abcam (1 mentions)
Anti fadd, by Abcam (1 mentions)
Anti ripk1, by BD (1 mentions)
Cycloheximide ch, by Merck Group (1 mentions)
Gsk 963, by GlaxoSmithKline (1 mentions)
Anti c flip, by Cell Signaling Technology (1 mentions)
Smac007, by GlaxoSmithKline (1 mentions)
Z vad fmk, by Enzo Life Sciences (1 mentions)
Goat anti mouse igg, by Santa Cruz Biotechnology (1 mentions)
Goat anti rabbit igg conjugated to horseradish peroxidase, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by Cytiva (1 mentions)
4 protocols using flag tagged tnf
1
Antibody Cocktail for Cell Death Signaling
2
Immunoprecipitation of FLAG-tagged TNF Receptor
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The indicated cell lines were seeded in sterile 10-cm 2 dishes in complete medium at a density of 1.0 × 10 6 cells per plate. After 24 hours, cells were serum-starved for 24 hours and incubated with FLAGtagged TNF (Enzo Life Sciences) at 1 g/ml at 4°C in serum-free medium for 30 min. Cells were washed twice with ice-cold PBS and lysed in lysis buffer [30 mM tris-HCl (pH 7.4), 150 mM NaCl, 1% (v/v) Triton X-100, and 10% (v/v) glycerol], supplemented with complete protease inhibitors (Roche). Lysates were incubated on ice for 20 min and centrifuged at 16,060g at 4°C for 20 min. Cleared lysates were incubated with DMP-cross-linked Protein G magnetic Dynabeads for 24 hours at 4°C and washed three times with lysis buffer. Immunoprecipitates were boiled in 2× Laemmli Sample Buffer [4% (w/v) SDS, 20% (v/v) glycerol, 120 mM tris-HCl (pH 6.8), and 0.02% bromophenol blue] and resolved by SDS-PAGE. Immunoprecipitated proteins were detected by immunoblotting with the appropriate antibodies and goat anti-mouse IgG or goat anti-rabbit IgG conjugated to horseradish peroxidase (Santa Cruz Biotechnology) and by enhanced chemiluminescence (Amersham).
3
Immunoprecipitation of TNF Receptor Complex
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Immunoprecipitation of the TNF receptor signalling complex (TNF‐RSC) was performed using 100 ng/ml FLAG‐tagged TNF (Enzo Life Sciences, Farmingdale, NY) as previously described (Fiil et al, 2013 ). For further details, see Appendix Supplementary Methods .
4
Characterizing TNF-TNFR1 Binding Modulation
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The effect of ABY TNFR1-1 on the TNF-TNFR1 interaction was determined by a pull-down assay with anti-Flag magnetic beads. HEK293 cells with endogenous expression of TNFR1 were lysed with native lysis buffer and protein concentration was determined using BCA. Ten μL of anti-FLAG magnetic beads were incubated with 30 μL of 25 μg/mL FLAG-tagged TNF (ALX-522-008-C050, Enzo Life Sciences) for two hours at 4 °C. Unbound TNF was then removed using magnet, and the anti-FLAG beads were washed three times with 0.1% PBSA. TNF coated beads were incubated with 250 μl of HEK293 lysate (7.5 mg/ml) with and without ABY TNFR1-1 , incubated overnight, and washed three times with PBSA. 10 μL of 1x loading dye (1610747, Bio-Rad) was added to the 10 μL of beads and pipetted up and down 5 times to elute the proteins. Using a magnet, the 10 μL of dye was then removed and placed in a separate tube. This elution step was repeated three more times for each sample for a total of 40 μL of loading dye per sample. Pulled-down samples were resolved by SDS-PAGE and immunoblotted with anti-Flag and anti-TNFR1 antibodies.
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