293T cells transfected with FLAG-tagged wild-type NT5C2 and HA-tagged NT5C2 R367Q were collected in lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 1 mM EDTA, 5% Glycerol, 5 mM β-ME, 0.1 % Triton). We incubated cell lysates with EZview™ Red ANTI-FLAG® M2 Affinity Gel clone M2 beads (Sigma) or EZview™ Red Anti-HA Affinity Gel beads (Sigma) for 2 hours at 4°C. Following four washes with lysis buffer and one wash with PBS, we boiled the beads in 2x SDS-loading buffer, separated them by SDS PAGE and transfered them to a nitrocellulose membrane for western blot analysis. We detected Flag-tagged proteins by immunoblot with a DYKDDDDK Tag Antibody (Cell Signaling Technology Cat # 2368) and HA-tagged proteins with an Anti-HA High Affinity antibody (Roche Cat #11867431001).
Dykddddk tag antibody
Manufactured by Cell Signaling Technology
Sourced in United States
The DYKDDDDK Tag Antibody is a monoclonal antibody designed to detect the DYKDDDDK peptide tag, which is commonly used for recombinant protein expression and purification. This antibody can be used to identify and track proteins of interest that have been engineered to contain the DYKDDDDK tag.
Automatically generated - may contain errors
Lab products found in correlation
Anti ha high affinity antibody, by Roche (1 mentions)
Ezview red anti ha affinity gel beads, by Merck Group (1 mentions)
Na931 1ml, by Cytiva (1 mentions)
6xhis monoclonal antibody albumin free, by Takara Bio (1 mentions)
Lipofectamine 2000, by Thermo Fisher Scientific (1 mentions)
Protein a g plus agarose, by Santa Cruz Biotechnology (1 mentions)
γ 32p atp, by PerkinElmer (1 mentions)
Trizol, by Thermo Fisher Scientific (1 mentions)
Anti β actin antibody, by Cell Signaling Technology (1 mentions)
Acetylated lysine antibody, by Cell Signaling Technology (1 mentions)
Phospho ampk thr 172 antibody, by Cell Signaling Technology (1 mentions)
4 protocols using dykddddk tag antibody
1
Purification and Detection of NT5C2 Proteins
2
Yeast Protein Isolation and Western Blot
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Total protein was isolated from mid-exponentially growing yeast by rapid capture of protein expression through 5% tricarboxylic acid treatment for 10 min, followed by a wash in acetonitrile. The cell pellets were then dried at room temperature for 30 min before bead-beating in Tris-acetate-EDTA buffer for 5 min at room temperature. Samples were then resuspended in SDS loading buffer from NuPage, boiled for 5 min, and loaded on 4–12% polyacrylamide Bis-Tris gels and separated by electrophoresis in MOPS buffer. Proteins were then transferred to a nitrocellulose membrane and were blocked for 1 h in TBST (Tris-buffered saline, 0.1% Tween 20) with 5% bovine serum albumin. Primary antibodies (DYKDDDDK Tag Antibody, Cell Signaling Technology 2368S; α-Pab1 Antibodies-Online ABIN1580454 (clone 1G1)) were incubated with membranes at a 1:1,000 dilution in TBST for 1 h at room temperature, washed with TBST, then incubated for 30 min at room temperature with anti-rabbit (Cell Signaling Technology, 7074S) and anti-mouse (Cytiva NA931-1ML) HRP-linked antibodies at a 1:10,000 dilution. Membranes were developed with Pierce ECL western blotting substrate and imaged on the chemiluminescence channel on a ProteinSimple instrument.
3
Fluorescent Labeling of Cascade Complex
4
AMPK Activation and Signaling Assay
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5-Aminoimidazole-4-carboxyamide ribonucleoside (AICAR) was from Toronto Research Chemicals (Downsview, ON, Canada). Adenosine 5´-monophosphate sodium salt (5´AMP), bTSH, threo-1, 4-Dimercapto-2, 3-butanediol (DTT) and were purchased from Sigma (St. Louis, MO). RIPA lysis buffer and bicinchoninic acid (BCA) protein assay kits were from Shen-neng Bo Cai (Shanghai,China), p-Ser and p-Thr antibodies, immunoglobulin IgG, SREBP-2 antibody, and protein A/G plus agarose were obtained from Santa Cruz Biotechnology (Santa Cruz, CA). Mouse LMNB1 monoclonal antibody was from Proteintech (ProteinTech Group, Chicago, IL, USA). [γ-32P]ATP (specific activity 3000Ci/mmol) was from Perkin Elmer (San Jose, CA, USA). 10×Assay buffer, Adenosine-5'-triphosphate (ATP), Acetylated-Lysine Antibody, DYKDDDDK Tag Antibody, phospho-Ser79 ACC1, anti-β-actin antibody, Rabbit polyclonal AMPK antibody and phospho-AMPK (Thr-172) antibody were purchased from Cell Signaling Technology (Beverly, MA), and rabbit polyclonal AMPK antibodies recognize the subunit α1 or α2 isoform. SAMS peptide (HMRSAMSGLHLVKRR) was purchased from Upstate Biotechnology (Lake Placid, NY). P81 phosphocellular paper was from GE healthcare (Piscataway, NJ). Trizol and Lipofectamine 2000 were purchased from Invitrogen (Carlsbad, CA).
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