The recombinant DUBs were captured with anti-AGIA tag-conjugated magnetic beads, using the same procedure as in the in vitro DUB assay. The recombinant DUB on the magnetic beads was combined with 10 µL of substrate mixture containing 2 µM of substrate ubiquitins shown in Figure S5 , in 50 mM Tris-HCl, pH 7.5, 5 mM DTT, and the deubiquitination assay was performed for 3 h at 30 °C. The supernatant was separated from the recombinant DUB on the beads by a magnetic stand, and a 5 µL portion of each reaction was transferred to a 384-well OptiPlate (PerkinElmer, Waltham, MA, USA) containing AlphaScreen beads mix (100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 1 mg/mL BSA, 0.1% Tween 20), 7.5 ng anti-DYKDDDDK (FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan), 0.08 µL streptavidin-conjugated donor beads, and 0.08 µL protein A-coated acceptor beads (PerkinElmer), in a total volume of 25 µL. The OptiPlate was incubated for 1 h at room temperature, and the luminescent signal was detected by an EnVision plate reader (PerkinElmer).
Anti dykddddk
Manufactured by Fujifilm
Sourced in Japan
Anti-DYKDDDDK is a lab equipment product manufactured by Fujifilm. It is designed to detect and isolate proteins tagged with the DYKDDDDK peptide sequence, which is a commonly used epitope tag. The core function of Anti-DYKDDDDK is to facilitate the purification and analysis of tagged proteins through affinity-based techniques.
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Lab products found in correlation
Pierce bca protein assay kit, by Thermo Fisher Scientific (2 mentions)
Protein a coated acceptor beads, by PerkinElmer (1 mentions)
Optiplate, by PerkinElmer (1 mentions)
Envision plate reader, by PerkinElmer (1 mentions)
Pd minitrap g 25 column, by Cytiva (1 mentions)
Complete edta free protease inhibitor, by Roche (1 mentions)
Anti phospho p70s6k, by Cell Signaling Technology (1 mentions)
Anti gfp, by Nacalai Tesque (1 mentions)
Anti spc1, by Merck Group (1 mentions)
Anti ha, by Roche (1 mentions)
Anti myc, by Fortrea (1 mentions)
Anti p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Immobilon p, by Merck Group (1 mentions)
Anti rhoa, by Santa Cruz Biotechnology (1 mentions)
Anti rac1, by BD (1 mentions)
Anti kras, by Santa Cruz Biotechnology (1 mentions)
Anti phospho p44 42 mapk erk1 2, by Cell Signaling Technology (1 mentions)
Anti rabbit igg hrp linked antibody, by Cell Signaling Technology (1 mentions)
Anti gapdh, by Proteintech (1 mentions)
Chemidoc, by Bio-Rad (1 mentions)
5 protocols using anti dykddddk
1
Deubiquitinase Activity Assay
2
Dye-conjugated Fab Preparation Protocol
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Dye-conjugated Fabs were prepared as previously described (Kimura and Yamagata, 2015 ). In brief, 100 μg purified Fab (anti-H3K9ac: CMA310/19E5, Hayashi-Takanaka et al., 2011 (link); anti-H3S28ph: CMA315/10-20F11, Hayashi-Takanaka et al., 2014 (link); anti-DYKDDDDK tag: Fujifilm Wako, 012-22384) was reacted with N-hydroxysuccinimide ester-conjugated fluorescence dye (Cy5, cytiva, PF11A25001; Cy3, cytiva, PA13105) in 100 mM NaHCO3 (pH 8.3) in PBS for 1 h at room temperature in the dark. Dye-conjugated Fabs were collected using PD MiniTrap G-25 column (Cytiva, 28918004; pre-equilibrated with PBS) and were concentrated to 1 mg/ml using a 10-kDa cutoff Amicon Ultracell Centrifuge Filter Unit (Merck, UFC5010BK), and stored at 4°C in the dark.
3
Affinity Purification of Membrane Proteins
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Rsv4-FLAG and P3-Myc were co-expressed in N. benthamiana leaves by agroinfiltration. 3 days after infiltration, the leaves were homogenized in TR buffer (30 mM HEPES-KOH pH 7.4, 80 mM KOAc, 1.8 mM Mg(OAc)2, 2 mM DTT, 1 tablet per 50 ml cOmplete EDTA-free Protease Inhibitor [Roche]). After clarification by centrifugation (800×g), membranes were pelleted by 16,000×g for 15 min. The pellet was suspended in TR buffer containing 1% Triton X-100, incubated with anti-FLAG antibody at 4 °C for 2 h, washed four times with wash buffer (30 mM HEPES-KOH pH 7.4, 100 mM NaCl, 2 mM DTT, 0.2% Triton X-100, 1 tablet per 50 ml cOmplete EDTA-free Protease Inhibitor [Roche]), and eluted with wash buffer containing 0.1 mg per ml 3× FLAG peptide. The eluted samples were analyzed by western blotting using anti-Myc (Cat#16286-1-AP: Cosmo Bio, Tokyo, Japan) and anti-DYKDDDDK (Cat#018-22381: Wako Pure Chemical, Osaka, Japan) antibodies diluted at 1:1000 in TBST containing 5% skim milk.
4
Protein Extraction and Detection
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Cells grown in YES medium were mixed with trichloroacetic acid at a final concentration of 10% and harvested. Collected cell pellets were then suspended in 10% trichloroacetic acid and vortexed vigorously with 0.5-mm-diameter zirconia beads for 5 min at 4°C. After breakage, cell suspensions were centrifuged at 800 × g for 10 min, and the supernatant was discarded. The remaining pellets were suspended in the SDS sample buffer containing 0.5 M Tris-HCl (pH 8.0) and boiled for 5 min, followed by centrifugation at 17,700 × g for 15 min. The recovered supernatant was separated by SDS-PAGE, transferred to nitrocellulose membrane, and probed with primary antibodies listed as follows; anti-phospho-p70 S6K (1:5000; Cell Signaling Technology) for phosoho-Psk1 (Thr-415) detection, anti-Spc1 (1:10000),83 (link) anti-FLAG (1:5000; Sigma-Aldrich) and anti-DYKDDDDK (1:5000; FUJIFILM Wako) for the FLAG-tagged protein detection, anti-myc (1:5000; Covance), anti-HA (1:2000; Roche) and anti-GFP (1:2500; Nacalai Tesque). Anti-rabbit IgG (H + L) HRP-conjugated (1:10000; Promega), anti-mouse IgG (H + L) HRP-conjugated (1:10000; Promega), and anti-rat IgG (H + L) HRP-conjugated (1:10000; Jackson ImmunoResearch) were used as secondary antibodies.
5
Western Blotting of Ras Pathway
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Cells were harvested and lysed in TNE buffer (20 mM Tris–HCl, pH 8.0, 150 mM NaCl, 2 mM EDTA, 1% NP-40) containing phosphatase inhibitors (10 mM NaF, 1.5 mM Na3VO4) and a protease inhibitor (1 mM PMSF) on ice. The protein concentration was determined using a Pierce BCA Protein Assay Kit (Thermo Fisher Scientific). Equal amounts of protein (15 μg protein) were separated by 12.5% SDS-PAGE and transferred by electrophoresis onto polyvinylidene difluoride membranes (Immobilon-P, Millipore). Membranes were incubated overnight at 4 °C with the indicated antibodies diluted to 1:4000 (anti-K-Ras [Santa Cruz, #sc-30], anti-phospho-p44/42 MAPK [Erk1/2]) (Cell Signaling Technology, #4370), anti-p44/42 MAPK [Erk1/2] (Cell Signaling Technology, #4695), anti-DYKDDDDK (FUJIFILM Wako Pure Chemical Corporation, 014-22383), anti-RhoA (Santa Cruz, #sc-418), anti-Rac1 [BD Transduction Laboratories, 610650, and anti-GAPDH (Proteintech, 60004-1-Ig]). After three washes with Tris-buffered saline containing 0.05% Tween 20, membranes were incubated with horseradish peroxidase (HRP)conjugated secondary antibodies (antimouse IgG HRP-linked antibody [Cell Signaling Technology, #7076], anti-rabbit IgG HRP-linked antibody [Cell Signaling Technology, #7074]), and chemiluminescence was detected using Chemidoc (Bio-Rad).
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