Myc-TEAD1 and Flag-YAP or Flag-VGLL4 were transfected into HEK293A cells and 24 – 48 h post-transfection, cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, PhosSTOP phosphatase inhibitor cocktail, cOmplete EDTA-free protease inhibitor). Anti-c-myc acceptor beads (Perkin Elmer) were added to each well and incubated for 2 h prior to addition of anti-FLAG donor beads (Perkin Elmer). Samples were incubated overnight in darkness and Alpha signals were recorded using Perkin Elmer EnVision plate reader.
Anti flag donor beads
Manufactured by PerkinElmer
Anti-FLAG donor beads are a type of laboratory equipment used for protein purification. They are designed to bind to proteins that have been tagged with a specific FLAG peptide sequence. These beads can be used to isolate and purify FLAG-tagged proteins from complex biological samples.
Automatically generated - may contain errors
Lab products found in correlation
Anti c myc acceptor beads, by PerkinElmer (3 mentions)
Envision plate reader, by PerkinElmer (2 mentions)
Enspire reader, by PerkinElmer (2 mentions)
Anti his acceptor beads, by PerkinElmer (2 mentions)
Alphaplate 384, by PerkinElmer (1 mentions)
Glutathione acceptor beads, by PerkinElmer (1 mentions)
6 protocols using anti flag donor beads
1
Protein-Protein Interaction Analysis
2
Claudin-4 Binding Assay with C-CPE
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The purified claudin-4 samples were diluted to 40 nM (final 10 nM) in the reaction buffer [50 mM Tris-HCl buffer (pH 7.0), containing 0.05% βDDM, 0.002% CHS, and 400 mM NaCl], and 5 μl aliquots were dispensed per well in a 384-well plate (AlphaPlate-384, PerkinElmer) on ice. In the blank well, an equal volume of the reaction buffer was dispensed. The purified GST-tagged C-CPE was also diluted to 160 nM (final 40 nM) in the reaction buffer, and a 5 μl aliquot of diluted GST-tagged C-CPE was added to all wells, mixed by shaking 30 sec, and then incubated on ice. Two hours later, a 5 μl aliquot of 80 μg/ml (final 20 μg/ml) Glutathione acceptor beads (PerkinElmer) in the reaction buffer was added to all wells, mixed by shaking 30 sec, and then incubated on ice for 1 hr in the dark. Finally, a 5 μl aliquot of 80 μg/ml (final 20 μg/ml) Anti-FLAG donor beads (PerkinElmer) in the reaction buffer was added to all wells, mixed by shaking 30 sec, and then incubated on ice for 1 hr in the dark. Before measurement, the plate was incubated at room temperature for 20 min in the dark. Alpha counts were measured by EnVision (PerkinElmer), using the standard AlphaScreen settings.
3
Protein-Protein Interaction Assay
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Example 3
Myc-TEAD1 and Flag-YAP or Flag-VGLL4 were transfected into HEK293A cells and 24-48 h post-transfection, cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, PhosSTOP phosphatase inhibitor cocktail, cOmplete EDTA-free protease inhibitor). Anti-c-myc acceptor beads (Perkin Elmer) were added to each well and incubated for 2 h prior to addition of anti-FLAG donor beads (Perkin Elmer). Samples were incubated overnight in darkness and Alpha signals were recorded using Perkin Elmer EnVision plate reader.
4
Protein-Protein Interaction Analysis
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Myc-TEAD1 and Flag-YAP or Flag-VGLL4 were transfected into HEK293A cells and 24 – 48 h post-transfection, cells were lysed with lysis buffer (20 mM Tris-HCl, pH 7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1% Triton X-100, PhosSTOP phosphatase inhibitor cocktail, cOmplete EDTA-free protease inhibitor). Anti-c-myc acceptor beads (Perkin Elmer) were added to each well and incubated for 2 h prior to addition of anti-FLAG donor beads (Perkin Elmer). Samples were incubated overnight in darkness and Alpha signals were recorded using Perkin Elmer EnVision plate reader.
5
Inhibition of Mouse Pol κ-REV1 CTD
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Example 15
The FLAG-tagged mouse POL κ-REV1 CTD was diluted in PBS containing 1 mM Tris(2-carboxyethyl)phosphine (TCEP) and 0.005% Tween-20 at a final protein concentration of 1 nM and transferred to individual wells of a 96-well, half-area, white opaque plate (PerkinElmer). Serially diluted RE01 stock solutions in 50% DMSO were added to the wells to yield final inhibitor concentrations of 0-25 μM in 2% DMSO. After 30 min incubation, anti-FLAG Donor Beads (PerkinElmer) were added to a final concentration of 20 ng/μL to individual wells and incubated for an hour. His8-tagged mouse REV7/3 was subsequently added to the reaction mixture to a final concentration of 10 nM and incubated for 30 min. Anti-His Acceptor Beads (PerkinElmer) were added to a final concentration of 20 ng/μL and incubated for an hour. The chemiluminescent signals were observed with a PerkinElmer Enspire Reader at the excitation wavelength of 680 nm and detection wavelength of 615 nm.
Fitting of the inhibition curve (
6
Inhibition Assay of Mouse POL κ-REV1
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