Recombinant Flag-tagged RNF114 was used as bait to precipitate pure recombinant p21 (Origene Technologies Inc., TP309752 and TP720567) using Anti-Flag agarose beads (GenScript Biotech Corp., L00432). One microgram of Flag-RNF114 was added to 50 μL of TBS, followed by the addition of nimbolide (100 μM final concentration, Cayman Chemical Co., 19230) or equivalent volume of DMSO. Samples were incubated at room temperature for 30 min. One microgram of pure p21 was added to each sample, and samples were incubated at room temperature 30 min with agitation. Ten microliters of Flag agarose beads were added to each sample, and samples were agitated at room temperature for 30 min. Washes (3 times, 1 mL TBS) were performed before proteins were eluted using 50 μL of TBS supplemented with 250 ng/μL 3 × FLAG peptide (ApexBio A6001). Supernatant (30 μL) were collected and after the addition of Laemmli’s reducing agent (10 μL), samples were boiled at 95 °C for 5 min and allowed to cool. Samples were analyzed by Western blotting as described above.
Anti flag agarose beads
Manufactured by GenScript
Anti-Flag agarose beads are a laboratory product used for protein purification. They are composed of agarose beads that have been conjugated with anti-Flag antibodies. The core function of these beads is to bind and capture proteins that contain a Flag tag, allowing for their isolation and purification from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Flag peptide, by Apexbio (2 mentions)
Nimbolide, by Cayman Chemical (2 mentions)
Tp309752, by GenScript (2 mentions)
Tp720567, by GenScript (2 mentions)
Peroxidase conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
Protease inhibitor, by Merck Group (1 mentions)
Chemidoc touch imaging system, by Bio-Rad (1 mentions)
Nitrocellulose membrane, by Cytiva (1 mentions)
Clarity western ecl substrate, by Bio-Rad (1 mentions)
3 protocols using anti flag agarose beads
1
Nimbolide Inhibits RNF114-p21 Interaction
2
Nimbolide Inhibits RNF114-p21 Interaction
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Recombinant Flag-tagged RNF114 was used as bait to precipitate pure recombinant p21 (Origene Technologies Inc., TP309752 and TP720567) using Anti-Flag agarose beads (GenScript Biotech Corp., L00432). One microgram of Flag-RNF114 was added to 50 μL of TBS, followed by the addition of nimbolide (100 μM final concentration, Cayman Chemical Co., 19230) or equivalent volume of DMSO. Samples were incubated at room temperature for 30 min. One microgram of pure p21 was added to each sample, and samples were incubated at room temperature 30 min with agitation. Ten microliters of Flag agarose beads were added to each sample, and samples were agitated at room temperature for 30 min. Washes (3 times, 1 mL TBS) were performed before proteins were eluted using 50 μL of TBS supplemented with 250 ng/μL 3 × FLAG peptide (ApexBio A6001). Supernatant (30 μL) were collected and after the addition of Laemmli’s reducing agent (10 μL), samples were boiled at 95 °C for 5 min and allowed to cool. Samples were analyzed by Western blotting as described above.
3
Co-Immunoprecipitation Protein Analysis
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For co-IP experiments, total lysates were prepared using lysis buffer (20 mM HEPES, pH 7.4, 100 mM NaCl, 2 mM DDM (n-dodecyl-β-D-maltoside, Anatrace), 0.02% CHS (cholesterol hemisuccinate, Sigma), protease inhibitors (Roche)). Then, the lysates were passed through a syringe needle for 15 times and incubated with agitation for 1 h at 4°C. The supernatants were prepared by centrifugation at 15,000 g for 10 min and incubated with anti-RFP agarose beads (MBL life science) or anti-Flag agarose beads (GenScript) for 4 h at 4 oC. The beads were washed three times with cold PBS. The protein were boiled in 2 × SDS running buffer and subjected to SDS-PAGE for western blotting.
For immunoblotting, proteins were separated by SDS-PAGE (Gensript) and transferred to the nitrocellulose membranes (Amersham). Then, the membranes was blocked for 1 h at room temperature with bovine serum albumin (BSA) in the blocking buffer (ABCONE), probed with specific primary antibodies overnight at 4 oC, and incubated with peroxidase-conjugated secondary antibodies (Jackson Immuno Research). The bands were visualized with chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) and imaged by a ChemiDoc Touch imaging system (Bio-Rad).
For immunoblotting, proteins were separated by SDS-PAGE (Gensript) and transferred to the nitrocellulose membranes (Amersham). Then, the membranes was blocked for 1 h at room temperature with bovine serum albumin (BSA) in the blocking buffer (ABCONE), probed with specific primary antibodies overnight at 4 oC, and incubated with peroxidase-conjugated secondary antibodies (Jackson Immuno Research). The bands were visualized with chemiluminescence (Clarity Western ECL Substrate, Bio-Rad) and imaged by a ChemiDoc Touch imaging system (Bio-Rad).
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