Ubiquilin 1 Tandem UBA (TUBE2) Agarose (Boston Biochem) was used to enrich polyubiquitylated proteins. Cells were treated with CZM, BTZ or DMSO for 8 hours and then lysed in buffer containing 40 mM Tris-HCl, pH7.6, 5 mM NEM, 0.1% NP-40 and bound to TUBE2 agarose at 4°C for 2 hours. Beads were washed three times with 40 mM Tris-HCl, pH7.6, 150 mM NaCl, 0.1% NP-40. Bound proteins were eluted with 2X SDS sample buffer and loaded onto 4–12% Tris-Glycine precast gel (Invitrogen). After transfer, the member was blotted with ubiquitin or AMOT antibodies (Supplementary Table 5 ).
Ubiquilin 1 tandem uba tube2 agarose
Manufactured by R&D Systems
Ubiquilin 1 Tandem UBA (TUBE2) Agarose is a laboratory reagent used for the purification and detection of polyubiquitinated proteins. It contains agarose beads conjugated with recombinant Ubiquilin 1 protein, which binds to polyubiquitinated proteins. This product can be used in various applications involving the study of protein ubiquitination and degradation.
Automatically generated - may contain errors
Lab products found in correlation
Laemmli buffer, by Bio-Rad (2 mentions)
Pierce ip lysis buffer, by Thermo Fisher Scientific (2 mentions)
Tris glycine precast gel, by Thermo Fisher Scientific (1 mentions)
Mg132, by Peptide Institute (1 mentions)
N ethylmaleimide, by Thermo Fisher Scientific (1 mentions)
Sample buffer solution with reducing reagent 6 for sds page, by Nacalai Tesque (1 mentions)
Bcl 2, by Agilent Technologies (1 mentions)
Phospho bcl 2, by Cell Signaling Technology (1 mentions)
Phospho erk1 2, by Cell Signaling Technology (1 mentions)
Bcl xl, by Cell Signaling Technology (1 mentions)
Erk1 2, by Cell Signaling Technology (1 mentions)
Bca analysis, by Thermo Fisher Scientific (1 mentions)
Gapdh, by Merck Group (1 mentions)
5 protocols using ubiquilin 1 tandem uba tube2 agarose
1
Enriching Polyubiquitylated Proteins Using TUBE2
2
Assessing CK1α Ubiquitination in KG-1 Cells
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For assessment of endogenous ubiquitination of CK1α 2 × 107 KG-1 cells were treated with DMSO, 1 or 10 μM lenalidomide for 4 hours and then lysed in IP lysis buffer containing 10 mM NEM and 10 μM MG132. Ubiquitinated proteins were pulled down by Ubiquilin 1 Tandem UBA (TUBE2) Agarose (Boston Biochem) for 4 hours at 4 °C and washed 3x with IP lysis buffer. Protein was eluted by incubation with Laemmli buffer (Biorad) at 95 °C for 5 minutes, separated by SDS-PAGE, transferred to PVDF membrane and probed with anti-CK1α.
3
Assessing Endogenous Ubiquitination of PLZF and SALL4
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To assess endogenous ubiquitination of PLZF and SALL4, 2 × 107 lt-NES cells were pretreated with DMSO or 1 μM MLN4924, treated with DMSO or 10 μM pomalidomide for 7 h, and then lysed with Pierce IP Lysis Buffer (ThermoFisher Scientific) containing 10 mM N-ethylmaleimide (ThermoFisher Scientific), 10 μM PR-619 (Nacalai Tesque), 10 μM MG132 (Peptide Institute), and protease/phosphatase inhibitor cocktail. Ubiquitinated proteins were pulled down by incubating lysates with Ubiquilin 1 tandem UBA (TUBE2) agarose (Boston Biochem) for 2 h at 4 °C and washing the beads 3 times with IP lysis buffer. Bound proteins were eluted by incubating the beads with Sample Buffer Solution with Reducing Reagent (6×) for SDS-PAGE (Nacalai Tesque) at 95 °C for 5 min and separated by SDS-PAGE, followed by immunoblotting using antibodies against PLZF, SALL4, and Ubiquitin.
4
Immunoblotting and Ubiquitination Analysis
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As described previously, cells were lysed, sonicated and protein concentration was quantified by BCA analysis (Thermo Scientific, Rockford, IL). 50μg of protein lysate was resolved on SDS-PAGE, transferred to PVDF membrane, and blotted with specific primary and secondary antibodies (Sigma-Aldrich) accordingly. [15 (link)] For IP-Western, 500μg of protein lysate was incubated with Ubiquilin 1 tandem UBA (TUBE2) agarose (Boston Biochem, Cambridge, MA) in the IP buffer (25mM Tris•HCl, pH7.4, 150mM NaCl and 1% NP-40) on a rotation platform at 4ºC overnight. Ubiquitinated proteins were bound to the agarose, washed three times with IP buffer and subjected to immunoblotting. [55 (link), 56 (link)] Primary antibodies were used for Mcl-1, CDK9, actin (Santa Cruz Biotechnology, Dallas, TX), phospho-Pol II CTD at Ser2 and Ser5, Pol II CTD (Covance, Princeton, NJ), phospho-ERK1/2, ERK1/2, phospho-CDK9, phospho-Bcl-2, Bcl-xL (Cell Signaling Technology, Danvers, MA), Bcl-2 (Dako, Carpinteria, CA) and GAPDH (EMD Millipore, Billerica, MA). Densitometry of immunoreactive bands on immunoblots was measured with FluorChem E System (ProteinSimple, Santa Clara, CA), quantified and normalized with AlphaView (ProteinSimple).
5
Assessing CK1α Ubiquitination in KG-1 Cells
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