Substrate proteins and ubiquitination enzymes were prepared as described above. Ubiquitination reactions were set up in reaction buffer (50 mM HEPES pH 8.0, 150 mM NaCl, 5 mM MgCl2, and 5% glycerol) in 20 μL aliquots as follows: 5 μM Uba1 (E1), 5 μM Ubc4 (E2), 5 μM Rsp5 (E3), 20 μM substrate, 500 μM methylated ubiquitin (Millipore), 5 mM ATP and incubated in a thermocycler for 3 hours at 25°C. 24 individual 20 μL reactions were performed for a typical prep. After three hours, reactions were pooled and HRV3C-protease was added and allowed to cleave for 30 minutes at room temperature. Ubiquitination enzymes and His-MBP were removed by batch binding to MagneHis™ (Promega) magnetic Ni2+-NTA resin for 1 hour at 4°C. Resin was pelleted in a magnetic tube rack, and the supernatant was collected for gel based single-turnover ubiquitin-independent degradation assays.
Magnehis
Manufactured by Promega
MagneHis is a magnetic bead-based system for rapid and efficient purification of histidine-tagged proteins. It utilizes a highly specific interaction between the histidine-tag and the nickel-charged magnetic beads to capture and isolate the target protein from complex samples.
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Lab products found in correlation
3 protocols using magnehis
1
Ubiquitin-Dependent Degradation Assay
2
Ubiquitin Purification from Yeast
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In all experiments, about 1μl of MagneHis (Promega) was used per 200μg of protein extract. For HA-tagged substrate validation, 150ml of cells carrying His8-ubiquitin plasmid (BPM30)59 (link) or corresponding empty plasmid for control were grown in SD-URA media at 25°C. Cells with or without 20min heat-shock treatment at 45°C were washed twice with ice-cold 1xTBS and cell pellets snap frozen in liquid nitrogen. Thawed cells were lysed in HU buffer (8M urea, 100mM HEPES at pH38, 0.05% SDS, 10mM chloroacetamide, 1mM PMSF, 10mM imidazole and protease inhibitors cocktail) by glass beads. Following 90min incubation with cell extracts at ambient temperature, nickel beads were washed three times in HU buffer with 1% SDS. Bound proteins were eluted by incubating the beads in one volume of 8M HU and one volume of 2M imidazole for 10min at ambient temperature with shaking. One volume of 3×SDS-PAGE Laemmli sample buffer was added and samples were heated at 70°C for 10min before western blot analysis.
3
Affinity Purification of His-Tagged Proteins
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For immobilized metal ion affinity chromatography (IMAC), about 1 μl of MagneHis (Promega) was used per 200 μg of protein extract. A total of 150 ml of cells carrying H8-Ubi plasmid (BPM30)67 (link) or corresponding empty plasmid for control were grown in SC-URA media at 25 °C. Cells with or without 20 min HS treatment at 45 °C were washed twice with cold 1 × TBS and snap frozen in liquid nitrogen. Thawed cells were lysed in HU buffer (8M urea, 100 mM HEPES at pH 8, 0.05% SDS, 10 mM chloroacetamide, 1 mM PMSF, 10 mM imidazole and protease inhibitors cocktail) by glass beads. Following 90 min incubation with cell extracts at ambient temperature, nickel beads were washed three times in HU buffer with 1% SDS. Bound proteins were eluted by incubating the beads in one volume of 8M HU and one volume of 2M Imidazole for 10 min at ambient temperature with shaking. 1/3 volume of 3 × SDS–PAGE Laemmli sample buffer was added before heating elution at 70 °C for western blot analysis or in-gel digestion.
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