The reagents used were as follows: 3-Methyladenine (3 MA; 189490, Sigma), MG132 (474790, Millipore), Roscovitine (SC−24002, Santa Cruz), zymosan (Z4250, Sigma), lipopolysaccharide (LPS; L2880, Sigma), imiquimod (IMQ; HY-B0180A, MCE). Antibodies: RIG-I (3743, CST), MDA5 (5321, CST), CDK5 (ab40773, Abcam), CDK5 (AHZ0492, Invitrogen), P65 (39369, Active Motif), phospho-p65 (Ser529; 39691, Active Motif), phospho-TBK1 (Ser172; 5483, CST), TBK1 (3504, CST), GFP-tag antibody (M20004, Abmart), phospho-IRF3 (Ser396; 4947, CST), IRF3 (11904, CST), Flag-tag antibody (F1804, Sigma), MyD88 (4283, CST), β-actin (60008–1, Proteintech), phospho-Stat1 (Tyr701; 9167, CST), Stat1 (14994, CST), Flag-tag antibody (F7425, Sigma), β-tubulin (M20005, Abmart), phospho-Stat2 (Tyr690; 88410, CST), Stat2 (72604, CST), phospho-IRAK4 (Thr345/Ser346; 11927, CST), IRAK4 (4363, CST), GAPDH (60004–1-Ig, Proteintech), GFP (A) beads (KTSM1301, KT Health) and anti-Flag M2 beads (A2220, Sigma).
Flag tag antibody
Manufactured by Merck Group
Sourced in United States
The Flag-tag antibody is a laboratory tool used for protein detection and purification. It recognizes a specific amino acid sequence, known as the Flag-tag, which can be attached to proteins of interest. This antibody allows for the identification and isolation of the tagged proteins from complex mixtures.
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Lab products found in correlation
Sars cov 2 n protein antibody, by Sino Biological (2 mentions)
P eif2α, by Abcam (2 mentions)
Total pkr, by Abcam (2 mentions)
P pkr, by Abcam (2 mentions)
Protease inhibitor cocktail, by Merck Group (2 mentions)
Anti flag m2 bead, by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Zymosan, by Merck Group (1 mentions)
3 methyladenine, by Merck Group (1 mentions)
Roscovitine, by Santa Cruz Biotechnology (1 mentions)
β actin, by Proteintech (1 mentions)
β tubulin, by Abmart (1 mentions)
Gapdh, by Proteintech (1 mentions)
β tubulin antibody, by Merck Group (1 mentions)
Anti snap 25, by Synaptic Systems (1 mentions)
Protein a g agarose beads, by Merck Group (1 mentions)
Phenyl methyl sulfonyl fluoride (pmsf), by Merck Group (1 mentions)
Diphenyleneiodonium chloride, by Merck Group (1 mentions)
β actin, by Cell Signaling Technology (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
16 protocols using flag tag antibody
1
Immune Signaling Pathway Regulation
2
Antibody Sources for Protein Identification
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The HA-tag antibodies were purchased from Pierce, the FLAG-tag antibodies from SIGMA-ALDRICH and the anti-GFP antibodies from Thermo Scientific/Molecular Probes. The anti-SNAP-25, Stx1A, Stx1B, MAP2 and vGlut1 antibodies were purchased from Synaptic Systems, the β-Tubulin antibodies from SIGMA-ALDRICH and the VAPA antibodies from abcam. Antibodies against ORP1L and ORP2 have been characterized previously [20 (link), 51 (link)]. The anti Sec9p antibody was obtained from Dr. Patrick Brennwald (University of North Carolina at Chapel Hill, USA). The anti-Sso1/2p (K8) antibody [52 (link)] has been published previously. The anti-Hsp150p antibody [53 (link)] was a kind gift from Prof. Marja Makarow (University of Helsinki). The anti-Snc1p and Snc2p antibodies were obtained from Prof. Michael Knop (ZMBH of the University of Heidelberg, Heidelberg, Germany).
Alexa Fluor conjugated secondary antibodies for immunofluorescence staining were purchased from Jackson ImmunoResearch.
Alexa Fluor conjugated secondary antibodies for immunofluorescence staining were purchased from Jackson ImmunoResearch.
3
Protein Immunoblotting Analysis of SARS-CoV-2 Infection
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Confluent cell monolayers in a 12-well tissue culture plate were infected with the viruses and collected at the time points indicated in the related figure legends. SDS-PAGE, membrane transfer, antibody blotting, and protein band detection were performed as described previously (18 (link)). Total PKR, p-PKR, p-EIF2α, and actin antibodies were purchased from Abcam. FLAG tag antibodies were purchased from Sigma-Aldrich. VACV D12 protein antibody was described previously (27 (link)). SARS-CoV-2 N protein antibody was purchased from Sino Biological.
4
Protein Immunoblotting Analysis of SARS-CoV-2 Infection
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Confluent cell monolayers in a 12-well tissue culture plate were infected with the viruses and collected at the time points indicated in the related figure legends. SDS-PAGE, membrane transfer, antibody blotting, and protein band detection were performed as described previously (18 (link)). Total PKR, p-PKR, p-EIF2α, and actin antibodies were purchased from Abcam. FLAG tag antibodies were purchased from Sigma-Aldrich. VACV D12 protein antibody was described previously (27 (link)). SARS-CoV-2 N protein antibody was purchased from Sino Biological.
5
Identifying Interacting Proteins of USP41
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CoIP-MS was used to explore the interacting proteins with USP41. The process consists of 5 main steps:
(1) incubation of cell lysates with Flag-tag antibodies (Sigma-Aldrich, USA), following cell transfection with Flag-USP41 and cell lysis with RIPA lysate, (2) binding of immune complexes to protein A/G agarose beads (Sigma-Aldrich, USA), (3) removal of non-interacting proteins, (4) elution to obtain protein interacting complexes, (5)mass spectrometry identi cation of protein interacting complexes. The enriched co-immunoprecipitation products were analyzed by mass spectrometry. Peptides with scores b20 were removed, and higher scores meant a better degree of matching with the secondary atlas. Peptides were searched and compared qualitatively in UniPro. The UniquePep Count and the Cover Percent were also evaluated as auxiliary metrics for the nal identi cation results.
(1) incubation of cell lysates with Flag-tag antibodies (Sigma-Aldrich, USA), following cell transfection with Flag-USP41 and cell lysis with RIPA lysate, (2) binding of immune complexes to protein A/G agarose beads (Sigma-Aldrich, USA), (3) removal of non-interacting proteins, (4) elution to obtain protein interacting complexes, (5)mass spectrometry identi cation of protein interacting complexes. The enriched co-immunoprecipitation products were analyzed by mass spectrometry. Peptides with scores b20 were removed, and higher scores meant a better degree of matching with the secondary atlas. Peptides were searched and compared qualitatively in UniPro. The UniquePep Count and the Cover Percent were also evaluated as auxiliary metrics for the nal identi cation results.
6
Antibody Sourcing for STAT and Actin
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We obtained antibodies for STAT1 (#14994), STAT2 (#72604), TNIK (#32712), and β-actin (#4970) from Cell Signaling (Danvers, MA). The MX1 (#sc-271024) antibody was acquired from Santa Cruz Biotechnology (Dallas, TX). Sigma-Aldrich (St. Louis, MO) provided the following reagents: Protease inhibitor cocktail (#P8340), PMSF (#36978), NEM (#E3876), Diphenyleneiodonium chloride (D2926), and FLAG tag antibody (#F3165). The Lipofectamine 2000 transfection reagent (#11668027) was purchased from Thermo Fisher Scientific (Waltham, MA).
7
Quantifying PPK1 Expression in E. coli
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The Δppk1::Apr/PJL02-ppk1 strain grown overnight was diluted into fresh LB broth containing 0 mM, 0.25 mM, 0.5 mM, 1 mM, or 2 mM isopropyl-β-d -thiogalactopyranoside (IPTG) with shaking (180 rpm at 37°C). When the strain reached the logarithmic phase, the bacterial precipitate was collected at 12,000 rpm for Western blotting with 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The polyvinylidene difluoride (PVDF) membrane was blocked with 5% fat-free milk and incubated with FLAG tag antibody (Sigma, USA) for 1 h at room temperature. After washing with Tris-buffered saline–Tween (TBST) three times, the PVDF membrane was placed into a Tanon‐4200 imager for imaging to analyze the expression level of PPK1.
8
Cloning and Expression of AtUSP in Arabidopsis
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AtUSP was cloned from an Arabidopsis cDNA library by the polymerase chain reaction (PCR), as previously described (Bréhelin et al., 2000 (link); Park et al., 2009 (link)). After confirmation of the entire coding sequence, the full-length AtUSP cDNA was ligated into EcoRI/ClaI sites of the binary vector pCAMBIA1300, which has a FLAG-tag in the N-terminal region (Figure S1C ). Agrobacterium tumefaciens GV3101 was transformed with the plasmid and used to transfect Arabidopsis by the floral dip method (Clough and Bent, 1998 (link)). Transformants were selected on MS plates containing 50 μg/ml hygromycin (Duchefa). AtUSP expression was analyzed using western blot analysis with FLAG-tag antibody (Sigma).
9
DAPI and FLAG Tag Antibody Protocol
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4′,6-Diamidino-2-phenylindole (DAPI) and FLAG tag antibody were purchased from Sigma (St. Louis, MO, USA). Cytokine detection kits and BacLight Live/Dead staining kits were purchased from Invitrogen (Carlsbad, CA, USA). Polyphosphate (polyP) was purchased from Aladdin (Shanghai, China). Morpholinepropanesulfonic acid (MOPS) minimal medium was purchased from Teknova. The plasmids and bacterial strains used in this study are listed in Table S1 in the supplemental material.
10
ChIP-Seq of 3xFLAG-Hlf in LSK Cells
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WT LSK cells were infected with a pMX-GFP control or a pMX-3xFLAG-Hlf/Hlf-IRES-GFP virus and grown for 5 days in basal OP9 medium supplemented with 50 ng/mL SCF, 10 ng/mL IL-7, 10 ng/mL Flt3L, and 5 ng/mL IL-3 (all from Peprotech). Next, 2 × 107 cells were cross-linked in 1% formaldehyde, and nuclei were prepared and snap-frozen in a dry ice/isopropanol bath. The frozen nuclei were lysed, and chromatin was sonicated in a Bioruptor (Diagenode) before immunoprecipitating with a FLAG-tag antibody (F3165; Sigma-Aldrich). Next, cross-links were reversed, and DNA was purified using QIAGEN PCR clean-up columns. Sequencing libraries were prepared using the Illumina TruSeq ChIP sample preparation kit (IP-202-1012; Illumina) and sequenced on an Illumina HiSeq 2500. Further details along with data-processing regimens can be found in Supplemental Experimental Procedures .
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