To crosslink protein-RNA complexes, cells cultured in a 10 cm dish were washed with PBS twice and received 254 nm UV irradiation (1500 J/m2) before protein extraction. Cellular proteins were extracted with RIPA buffer [50 mM HEPES (pH 7.6), 150 mM NaCl, 0.5% Triton X-100, and 0.5% sodium cholate] with protease inhibitors (G6521; Promega). Anti-DYKDDDDK tag antibody magnetic beads (Fujifilm Wako Pure Chemical) were added to the lysate and incubated for 1 h at 4 °C. The magnetic beads were washed with TBST four times and further washed with TBST + 1 M NaCl. For western blotting analysis, washed beads were mixed with sodium dodecyl sulfate-containing sample buffer and heated at 95 °C. To retrieve RNA, beads were treated with RQ1 RNase-Free DNase (Promega), and after a TBST wash, they were incubated with protease K (1 h at 37 °C). Then, phenol-chloroform extraction and ethanol precipitation were performed.
Anti dykddddk tag antibody magnetic beads
Manufactured by Fujifilm
Anti-DYKDDDDK tag antibody magnetic beads are a laboratory tool used to detect and purify proteins tagged with the DYKDDDDK peptide sequence, also known as the FLAG tag. These magnetic beads are coated with an antibody that specifically binds to the FLAG tag, allowing for efficient capture and isolation of the tagged proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
G6521, by Promega (1 mentions)
Rq1 rnase free dnase, by Promega (1 mentions)
Pierce microplate bca protein assay kit reducing agent compatible, by Thermo Fisher Scientific (1 mentions)
Ni sepharose excel resin, by Cytiva (1 mentions)
Ni sepharose excel, by Cytiva (1 mentions)
Protease and phosphatase inhibitor cocktail, by Roche (1 mentions)
5 protocols using anti dykddddk tag antibody magnetic beads
1
Crosslink Protein-RNA Complexes and Analyze
2
Immunoprecipitation and Western Blot Analysis
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Anti-DYKDDDDK tag antibody magnetic beads (Wako Pure Chemical Industries) were used for IP. In brief, cell lysate samples were mixed with beads that had been prewashed in IP buffer (50-mM Tris-HCl, pH 7.5, 150-mM NaCl, 0.1% NP-40, 1-mM EDTA, pH 8.0, 0.25% gelatin, and 0.02% sodium azide), and incubated with rotation overnight at 4°C. The beads were separated with a magnetic separator and mixed with 4× SDS sample buffer (0.25 mol/L Tris-HCl, 8% SDS, 40% glycerol, and 0.02% bromophenol blue, pH 6.8), then boiled in a heat block at 95°C for 3 minutes to elute the proteins. For detection of the DYKDDDK tag fusion protein, Western blotting was performed as described previously. TrueBlot anti-mouse IgG horseradish peroxidase (1:1000; Rockland, Limerick, PA) was used as the secondary antibody to avoid interference of immunoprecipitated immunoglobulin heavy and light chains.
3
Recombinant LYPD1 Purification Protocol
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Recombinant LYPD1 was prepared using a modification from previously described methods [4 (link)]. Briefly, human LYPD1 gene with a FLAG sequence was inserted after the signal sequence and the deleted sequence of the C-terminal GPI-anchored region was inserted into the pcDNA3.1 vector (pFLAG-LYPD1). After a 48-h transfection with pFLAG-LYPD1, 293FT cells were lysed and FLAG-LYPD1 was immunoprecipitated using anti-DYKDDDDK tag antibody magnetic beads (Wako). Proteins were quantitated using a Pierce Microplate BCA Protein Assay Kit - Reducing Agent Compatible (Thermo Scientific) according to the manufacturer's instructions.
4
Protein Expression and Purification
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The cDNA fragments of human Alix, GFP, and human TSG101 were cloned into pCMV vector to obtain pCMV-Alix-His, pCMV-GFP-His, pCMV-TSG101-His, and pCMV-FLAG-TSG101, respectively. PCR-based deletion was performed to obtain pCMV-TSG-UEV-His and pCMV-FLAG-TSG-ΔUEV. The constructed pCMV vectors were transfected into 293T cells with polyethylenimine. Cell lysates were collected at 72 h posttransfection and clarified via centrifugation. The His-tagged proteins were purified using Ni Sepharose Excel (Cytiva) following the manufacturer’s protocol. The FLAG-tagged protein was purified with anti-DYKDDDDK-tag antibody magnetic beads (FujiFilm) according to the manufacturer’s protocol by using 500 μg/μL of FLAG peptide. The expression vector pET15b-RABV M-His was constructed by cloning RABV M into pET15b vector and used to transform BL21 competent cells. The protein expression was induced with 0.1 mM isopropyl-β-d -thiogalactopyranoside (IPTG) at 37°C for 5 h. Cell pellets were lysed by sonication in lysis buffer. Solubilized protein was purified by affinity chromatography using Ni Sepharose Excel resin (Cytiva).
5
Immunoprecipitation and Immunoblot Analysis
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Transfected HEK293T cells were lysed with buffer containing 50 mM Tris, pH 7.5, 5 mM EDTA, 150 mM NaCl, and 0.1% Triton X-100 with protease and phosphatase inhibitor cocktail (Roche, Nutley, NJ, United States). FLAG-tagged proteins were immunoprecipitated using anti-DYKDDDDK tag antibody magnetic beads (FUJIFILM). Whole cell lysates or immunoprecipitated proteins were subjected to immunoblot analyses as previously described (Inaguma et al., 2011 (link); Inaguma et al., 2013 (link)). In brief, whole cell lysates were prepared using 1x Sodium Dodecyl Sulfate (SDS) sample buffer, containing 50 mM Tris-HCl and 2% SDS. The SDS polyacrylamide gel electrophoresis was performed using polyacrylamide gel and separated proteins were transferred to a PVDF membrane. Antibody dilutions are summarized in Supplementary Table S2 . For sequential detection, antibody stripping buffer (0.1 M Glycine-HCl pH 2.5) was used. Signal intensity was measured by ImageJ software (National Institutes of Health, Bethesda, MD).
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