HEK293 cells were transfected with FLAG-tagged ZO-1 and GFP-tagged claudin-3. Cells were washed with ice-cold PBS and lysed with FLAG IP buffer [20 mM Tris-HCl (pH 7.5), 150 mM NaCl, 1% Triton X-100, and protease inhibitors]. Lysates were incubated with 10 μL of anti-DYKDDDDK mAb beads (Wako Pure Chemical Industries) for 2 h. The beads were washed with FLAG IP buffer, and bound proteins were eluted in a SDS sample buffer. Aliquots of the lysates and eluates were immunoblotted with anti-DYKDDDDK and anti-GFP antibodies.
Anti dykddddk mab beads
Manufactured by Fujifilm
Anti-DYKDDDDK mAb beads are a laboratory tool used for the detection and purification of DYKDDDDK-tagged proteins. The beads are coated with monoclonal antibodies specific to the DYKDDDDK epitope, allowing for efficient capture and isolation of tagged proteins from complex samples.
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Lab products found in correlation
4 protocols using anti dykddddk mab beads
1
FLAG-ZO-1 and GFP-Claudin-3 Coimmunoprecipitation
2
FLAG-tagged Protein Immunoprecipitation
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Cells were washed with ice-cold PBS and lysed with FLAG IP buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, and protease inhibitors). Lysates were incubated with 5 μl of anti-DYKDDDDK mAb beads (Wako Pure Chemical Industries) for 2 h. Beads were washed with FLAG IP buffer and bound proteins were eluted in SDS sample buffer. Aliquots of the lysate and eluate were immunoblotted with anti- DYKDDDDK, anti-HA and anti-GFP antibodies.
3
Apoptosis Induction and ROCK/Caspase Inhibition
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Cells were treated with 250 ng/ml anti-Fas antibody (SY-001) purchased from MBL (Nagoya, Japan) and 10 mg/ml cycloheximide purchased from Sigma (St Louis, MO) to induce apoptosis. To inhibit ROCK or pan-caspase activity during apoptosis, cells were also treated with 10 µM Y-27632 or 50 µM Z-VAD-FMK purchased from MBL (Nagoya, Japan). Cells were washed with PBS and lysed with IP buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, and protease inhibitors). Lysates were incubated with 10 μl of anti-DYKDDDDK mAb beads (Wako Pure Chemical Industries) for 2 h. Beads were washed with IP buffer and bound proteins were eluted in SDS sample buffer. Aliquots of the lysate and eluate were immunoblotted with anti-FLAG antibody and anti-GFP antibody. The signal intensity was quantified by using ImageJ.
4
Immunoprecipitation and Western Blot Analysis
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Cells were washed with PBS and lysed with IP buffer (20 mM Tris-HCl [pH 7.5], 150 mM NaCl, 1% Triton X-100, and protease inhibitors). Lysates were incubated with either 1 μg anti-HA mAb conjugated to Protein G Sepharose (GE Healthcare) or 5 μl of anti-DYKDDDDK mAb beads (Wako Pure Chemical Industries) for 2 h. Beads were washed with IP buffer and bound proteins were eluted in SDS sample buffer. Aliquots of the lysate and eluate were immunoblotted with anti-FLAG antibody (1:1000), anti-HA antibody (1:1000) and anti-GFP antibody (1:1000).
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