Cells were lysed at 4 °C in extraction buffer (0.1 M NaCl, 20 mM HEPES pH 7.5, 1 mM EDTA, 5 mM NaF, 1 mM dithiothreitol, 0.3% Triton X-100, 5% glycerol, 0.25 mM phenylmethylsulfonyl fluoride, and complete protease inhibitor cocktail and phosphatase inhibitors). Homogenates were cleared by centrifugation (12,000 g, 10 min). Coimmunoprecipitation was performed according to previous techniques55 (link). In vitro IP was performed using purified recombinant proteins instead of the cell lysate. The antibodies used in the study were as follows: Anti-Myc (Cell Signaling, #2276); Anti-DDK (Origene, TA50011); Anti-DJ-1(Santa Cruz, sc27006 and sc32874; Cell Signaling, 2134S); Anti-β-Actin, #4970); Anti-GAPDH (Santa Cruz, sc-32233); Anti-mATP5G1/2/3(abcam, ab180149); Anti-ATPB (abcam, ab14730), and Anti-Bcl-xL(Cell Signaling, #2764); Anti-Puromycin (3RH11) (Kerafast, EQ0001).
Anti ddk
Manufactured by OriGene
Sourced in United States
Anti-DDK is a monoclonal antibody that specifically recognizes the DDK (FLAG) epitope tag. The DDK tag is a commonly used protein expression tag that can be added to recombinant proteins to facilitate detection and purification. Anti-DDK is designed for use in various applications, such as immunoprecipitation, western blotting, and immunohistochemistry, to detect and analyze DDK-tagged proteins.
Automatically generated - may contain errors
Lab products found in correlation
Anti β actin, by Merck Group (3 mentions)
Anti dj 1, by Santa Cruz Biotechnology (1 mentions)
Anti β actin, by Santa Cruz Biotechnology (1 mentions)
Anti gapdh, by Santa Cruz Biotechnology (1 mentions)
Anti atpb, by Abcam (1 mentions)
Anti puromycin 3rh11, by Kerafast (1 mentions)
Anti myc, by Cell Signaling Technology (1 mentions)
Anti bcl xl, by Cell Signaling Technology (1 mentions)
Protease inhibitor and phosphatase inhibitor, by Merck Group (1 mentions)
Anti phospho mtor, by Cell Signaling Technology (1 mentions)
Anti akt, by Cell Signaling Technology (1 mentions)
Anti mtor, by Cell Signaling Technology (1 mentions)
Anti phospho akt, by Cell Signaling Technology (1 mentions)
Immunobilon western chemiluminescent hrp substrate, by Merck Group (1 mentions)
M per mammalian protein extraction reagent, by Thermo Fisher Scientific (1 mentions)
Anti actin, by Santa Cruz Biotechnology (1 mentions)
Anti e2f1, by Santa Cruz Biotechnology (1 mentions)
Enhanced chemiluminescence, by GE Healthcare (1 mentions)
Hrp linked secondary antibody, by GE Healthcare (1 mentions)
Pxi imaging system, by Syngene (1 mentions)
15 protocols using anti ddk
1
Coimmunoprecipitation of Protein Complexes
2
Western Blot Analysis of Protein Signaling
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Cell proteins were extracted using M-PER Mammalian Protein Extraction Reagent (Thermo Fisher Scientific, Waltham, MA, USA), supplemented with protease inhibitor and phosphatase inhibitor (Sigma, St. Louis, MO, USA). Western blots were prepared by standard procedures using anti-DDK (Origene Tech, Rockville, MD, USA), anti-actin (Santa Cruz, CA, USA), anti-AKT, anti-phospho-AKT, anti-mTOR, and anti-phospho-mTOR (Cell signaling Technology, Danvers, MA, USA). Immunoreactivity was detected by an Immunobilon Western Chemiluminescent HRP Substrate (Millipore, Billerica, MA, USA).
3
Western Blot Analysis of hCD36 and DDK
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The lentivirally infected cell lysates were prepared and analysed by Western blotting as previously described [19 (link)]. The following primary antibodies were used: anti-hCD36 (1: 20000 in TBST; kindly provided by Dr. Tandon, Bethesda, USA) and anti-DDK (1: 2000 in 5% BSA in TBST; Origene). Primary antibody binding was detected by anti-mouse secondary antibody (1:20000 in TBST; Dako).
4
Western Blot Analysis of E2F1 and DDK
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Cells were lysed and protein concentration was determined using the Bradford reagent. PVDF membrane was probed with primary and secondary antibodies before developing using the ECL Plus Western Blotting Substrate (Pierce, 32132). Antibodies used are as follows: anti-E2F1 (Santa Cruz, sc-193X), anti-DDK (Origene, TA50011), anti-βactin (Sigma, A1978), ECL anti-rabbit IgG HRP linked whole antibody (from donkey) (Amersham, NA934V) and anti-mouse IgG HRP linked whole antibody (from sheep) (Amersham, NA931V).
5
Western Blot Analysis of Sonic Hedgehog Protein
6
Immunoblotting of Epigenetic Regulators
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Immunoblotting was performed to detect protein expression using the Quick Western Kit–IRDye® 680RD (LI-COR Biosciences) following standard protocol. Primary antibodies used were: anti-SETD7 (a gift from Dr. Susanne Gräslund at SGC), anti-H3K4me1, anti-Histone H3, anti-NFE2L2, Anti-PPARGC1A (Abcam), anti-DDK (Origene), anti-SOD2 (Abcam). Membrane transfer was performed on iBlot 2 Gel Transfer Device with iBlot 2 Transfer stacks (PVDF) (Life Technologies). Imaging analysis was performed on the Odyssey CLx Infrared Imaging System using Image Studio Ver 3.1 (LI-COR Biosciences).
7
Characterization of AF20 Monoclonal Antibody
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AF20 monoclonal antibody (mAb) was produced and characterized as previously described [1 (link),3 (link),4 (link)]. The following antibodies were obtained commercially: anti-TFR1 (CD71) (Santa Cruz Biotechnology, Inc.; sc-32272), anti-HSP90 (EMD Millipore; 05–594), anti-N+/K+ ATPase (abcam, ab7671) and anti-DDK (Origene; TA100011). PNGase F was purchased from New England Biolabs (P0704S). Apo transferrin and holo transferrin were purchased from EMB Millipore (616395 and 616397).
8
Molecular Markers of Tight Junction Regulation
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Primary antibodies for Western blot include anti–NKA β1 subunit (Upstate, 05-382), anti-occludin (Invitrogen, 71-1500), anti–zo-1 (Invitrogen, 61-7300), anti–zo-2 (Invitrogen, 71-1400), anti-actin (MilliporeSigma, A2066), anti-GAPDH (MilliporeSigma, CB1-001), anti–NKA β2 subunit (Abcam, ab185210), anti-DDK (OriGene, TA50011-100), anti-MRCKα (Santa Cruz Biotechnology Inc., sc-374568), anti-MYPT1 (Cell Signaling Technology, 2634S), anti–phospho-MYPT1 (Thr696, Cell Signaling Technology, 5163S), anti-MLC2 (Cell Signaling Technology, 3672S), and anti–phospho-MLC2 (Ser19, Cell Signaling Technology, 3671S). The primary antibodies for immunofluorescence include anti–occludin Alexa Fluor594 (Invitrogen, 331594), anti–zo-1-Alexa Fluor594 (Invitrogen, 339194), and anti-MRCKα (Thermo Fisher Scientific, PA1-10038). The inhibitor for MRCKα BDP5290 was purchased from Aobious. Myosin inhibitor blebbistatin was purchased from Abcam.
9
Investigating Inflammatory Signaling in Cell Lines
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HEK293 and Caco-2 cells were purchased from the German Collection of Microorganisms and Cell Cultures (DSMZ). HEK293 cells were maintained in DMEM containing 10% (v/v) fetal calf serum (FCS). Caco-2 cells were maintained in MEM containing 20% (v/v) FCS. Cells were kept at 37°C with 5% CO2. Recombinant TNF-α was purchased from ImmunoTools, muramyl dipeptide (MurNAc-L-Ala-D-isoGln, MDP) from Bachem and diphenyleneiodonium chloride (DPI) from Sigma. p22phox (rb) antibody was purchased from Santa Cruz (sc-20781), NOD2 (rb, ms) antibodies from Novus Biologicals (rb: NB 500–253, ms: NB 100–524), Anti-Flag (ms) from Sigma (F1804), Anti-DDK (ms) from Origene (TA50011-100), Anti-GFP (rb) from BD (632377), Anti-IκBα (rb) from CST (2859) and Rabbit TrueBlot from Life Technologies (18-8816-33). All HRP-conjugated secondary antibodies were obtained from TH Geyer (ms: NA931V). AF-488-(A21206, A11055), AF-555-(A31570, A31572), and AF633-(A21082, A21052) conjugated secondary antibodies were purchased from Invitrogen. Transfections were performed using Fugene6 according to manufacturer's instructions (Roche).
10
Comprehensive Antibody Validation Protocol
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A GFP primary antibody (JL-8, Clontech) and an anti-HA (Y-11, Santa Cruz) were diluted to a 1∶10,000 to detect overexpressed protein samples. An anti-DDK (Origene) was used at a 1∶4,000 dilution to detect overexpressed hTTP-Flag and hZfp36l1-Flag. The membranes were incubated with a secondary antibody, goat anti-mouse IgG (Santa Cruz) or goat anti-rabbit (Bio-Rad), at a 1∶10,000 dilution, both antibodies were HRP-conjugated. To detect the endogenous ZFP36L2 protein, the anti-ZFP36L2 polyclonal antibody (C2-ZFP36L2-AS, described in [5] (link)) was used at a 1∶10,000 dilution followed by a secondary goat anti-rabbit at a 1∶10,000 dilution. As a loading control, a β-actin rabbit polyclonal antibody (N-21, Santa Cruz) was used at a 1∶5,000 dilution, followed by a secondary goat anti-rabbit at a 1∶10,000 dilution. The LHR antibody was used at a 1∶2,000 dilution (ProteinTech), followed by a secondary goat anti-rabbit at a 1∶10,000 dilution. All antibody signals were developed using a SuperSignal West Pico Chemiluminescent Substrate (Pierce) according to the manufacturer's instructions.
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