Ni-NTA Superflow Resin (Qaigen) was washed three times with Urea Buffer (10mM imidazole, 0.2% NP-40, 8 M urea, 500 mM NaH2PO4, 50 mM Tris HCl, pH 8.0). For each immunoprecipitation, 10 µl of resin was added per tube, and resuspended to 191 µl in Urea buffer. Extracts were supplemented with 100 µM of HIS6-Ubiquitin (Boston Biochem) and replication was carried out as described above. At the indicated time, 9 µl of extract was mixed with the bead mix and samples were incubated for one hour at room temperature, with end-over-end rotation. Resin was washed three times with urea buffer. All residual buffer was removed, and resin was boiled for 5 minutes in 30 μl sample buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 6.1% SDS, 0.01% bromophenol blue, 10% β-mercaptoethanol). 30 µl of 0.5 M imidazole was added to each sample and HIS6-tagged proteins were eluted off the resin for 60’ at room temperature, with gentle agitation. Resin was spun down at 1000 RCF for 1 minute, and the supernatant was removed. 10 µl of each sample was resolved on an SDS-PAGE gel alongside an input control and analyzed by Western blotting using the previously-described antibody against MCM749 (link). In extended Data Fig. 8D , a longer exposure of the IP lanes is shown, since they are far less intense than the input lanes.
His6 ubiquitin
Manufactured by R&D Systems
Sourced in United Kingdom
His6-ubiquitin is a recombinant protein that contains a hexahistidine (His6) tag fused to the N-terminus of ubiquitin. Ubiquitin is a small regulatory protein found in most tissues of eukaryotic organisms and plays a role in protein degradation and other cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Recombinant human ubiquitin, by R&D Systems (1 mentions)
Mastercycler nexus, by Eppendorf (1 mentions)
Flag peptide, by Merck Group (1 mentions)
M2 beads, by Merck Group (1 mentions)
Hsp60, by Abcam (1 mentions)
Oxphos cocktail, by Abcam (1 mentions)
Gfp trap, by Proteintech (1 mentions)
Actin hrp, by Merck Group (1 mentions)
Flag m2 affinity resin, by Merck Group (1 mentions)
Flag tag (flag), by Merck Group (1 mentions)
Anti flag antibody clone m2, by Merck Group (1 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Polyubiquitin chains, by R&D Systems (1 mentions)
His6 ube1, by R&D Systems (1 mentions)
His6 e1, by R&D Systems (1 mentions)
8 protocols using his6 ubiquitin
1
Analysis of Ubiquitinated Proteins by Ni-NTA Pulldown
2
Ubiquitin Conjugation Assay with LUBAC
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Reaction mixtures were prepared containing Ube1, UbcH7, and either LUBAC or HOIP, HOIL-1L, and SHARPIN with all proteins at 0.338 µM final concentration in a buffer containing 50 mM HEPES, 150 mM NaCl, 0.5 mM MgSO4, pH 8.0 (described in Ikeda et al., 2011 (link)). Reactions were started by addition of 59.0 µM ubiquitin or fluorescein-tagged ubiquitin, 2 mM ATP, and 0.5 µg of NEMO where indicated, then incubated at 37°C in a Mastercycler nexus (Eppendorf, Germany) thermocycler for 2 hr unless otherwise stated. Reaction was stopped by addition of SDS buffer and boiling at 95°C, proteins were resolved by SDS-PAGE, and analysed by immunoblotting with the indicated antibodies or by fluorescence imaging. Recombinant human ubiquitin, His6-ubiquitin, and ubiquitin KR mutants (K6R, K11R, K27R, K29R, K33R, K48R, K63R, K0) were purchased from Boston Biochem.
3
RRM1 Ubiquitination by RNF2 and Bmi1
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AD293 cells were transfected with Flag-RRM1, HA-RNF2, and GFP-Bmi1 and lysed after 48 h. Flag-RRM1 protein was immunoprecipitated with anti-Flag Ab cross-linked beads (M2-beads, Sigma), eluted by Flag-peptide (Sigma), and used as the substrate. HA-RNF2 and GFP-Bmi1 immunoprecipitated with anti-HA and anti-GFP Ab-conjugated agarose (Pierce) were used as E3 ligases. Flag-RRM1 (2.5 µL) and 20 µL anti-HA, anti-GFP IPs were incubated with 500 ng of E1 (Boston Biochem), 500 ng of E2 (UbcH5b and UbcH5c; Boston Biochem), and 5 µg His6-ubiquitin (Boston Biochem) in 50 µl reaction buffer (50 mM Tris [pH 7.5], 2.5 mM MgCl2, 15 mM KCl, 1 mM dithiothreitol, 0.01% Triton X-100, 1% glycerol, 8 mM ATP, 25 µM MG132, and protease inhibitors) at 37°C for 1 h. The reactions were terminated with SDS buffer containing β-mercaptoethanol and processed for 10% SDS-PAGE. Western blotting experiments were performed using the anti-ubiquitin antibody.
4
Protein Purification and Antibody Preparation
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A list of plasmids is provided in Table S1 . Plasmids were prepared by standard methods. Antibodies against the following proteins were purchased: UBQLN1/2 (Sigma, clone 5F5), UBQLN4 (Abcam ab106443), L9 and Tom20 (Santa Cruz Biotech., T-17 and FL-145), HA (Covance, clone 16B12), Rpt5 (Abcam ab22635), α7 (Enzo Life Sciences PW8110), FLAG (Sigma, clone M2), Hsc/Hsp70 (AssayDesigns, SPA-822), Hsp60 (Abcam ab46798), ClpP (Abcam ab124822), Actin-HRP (Sigma A3854), OxPhos cocktail (Abcam ab110411). FLAG-M2 Affinity resin was from Sigma, and GFP-trap from Chromotek. Antibodies to Bag6, TRC40, SGTA, TRAPα, GFP, and RFP have been described (Fons et al., 2003 (link), Mariappan et al., 2010 (link), Hessa et al., 2011 (link)). Anti-Myc was clone 9E10. Anti-HA used for IPs and blots (e.g., Figure 2 A) was raised in rabbits against the KLH-HA peptide conjugate. Recombinant proteins were either purchased (His6-Ubiquitin, E1, and E2 from Boston Biochem) or expressed and purified from E. coli as described (Mateja et al., 2015 (link)).
5
Ubiquitination Assay with DDX3X
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Recombinant His6-DDX3X (0.75 μM), TRIM25 (with His6-MBP-tag cleaved, 0.75 μM), Ube1-His6 (0.25 μM), His6-UbcH5b (0.75 μM) and His6-ubiquitin (5 μM, Boston Biochem, Cambridge, UK,) were incubated in a final volume of 20 μL in 1× ubiquitination reaction buffer (30 mM HEPES pH 7.4, 5 mM MgCl2·6H2O, 0.2 mM DTT) using a Thermomixer Comfort fitted with a 0.6 mL tube heating block (Eppendorf, Hamburg, Germany) set to 30 °C and 650 rpm for 1 h. Reactions were stopped by boiling with 2× Laemmli sample buffer prior to SDS-PAGE and immunoblotting. For inhibition experiments, His6-IAV-NS1 or His6-MBP (0.75–3.75 μM) were included in the reaction.
6
Antibody Panel for Ubiquitin Research
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Antibodies used in this study are as follows: anti‐Myc antibody (clone 9E10; Covance, Princeton, NJ), anti‐Flag antibody (clone M2; Sigma, St. Louis, MO), anti‐HA antibody (HA.11 clone 16B12, Covance, Princeton, NJ), anti‐Alpha‐tubulin antibody (ab15246, Abcam, Cambridge, UK), anti‐Linear Ub antibody (LUB9; Life Sensors, Malvern, PA), anti‐Lys 63‐Ub antibody (Apu3; Merck Millipore, Darmstadt, Germany), anti‐Lys 48‐Ub antibody (Apu2; Merck Millipore), and anti‐Ub antibody (clone P4D1; Santa Cruz Biotechnology, Santa Cruz, CA). A rabbit polyclonal antibody recognizing LUBEL‐RBR was generated using recombinant LUBEL‐RBR (aa 2,514–aa 2,655) (immunoGlobe, Himmelstadt, Germany). His6 Ube1, ubiquitin, His6‐ubiquitin, di‐ubiquitin chains (Lys 6, Lys 11, Lys 27, Lys 29, Lys 33, Lys 48, Lys 63‐linked and linear chains), poly‐ubiquitin chains (2–7, Lys 48 and Lys 63‐linked chains, and tetra linear chains) were purchased from Boston Biochem (Cambridge, MA).
7
In vitro ubiquitination assay for FLS2
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The in vitro ubiquitination assay was performed as described with some modifications (Lu et al., 2011 (link)). The reactions contain 1 μg of purified MBP–FLS2CD, 1 μg of purified His6-E1 (AtUBA1), 1 μg of purified His6-E2 (AtUBC8), 1 μg of His6-ubiquitin (Boston Biochem), 1 μg of purified GST–PUB, and 1–16 μg of purified GST or GST–ARM with 6 μl of 5× ubiquitination buffer (0.1M TRIS-HCl, pH 7.5, 25mM MgCl2, 2.5mM DTT, 10mM ATP) to a final volume of 30 μl. The reactions were incubated at 30 °C for 2h, and then stopped by adding SDS sample buffer and boiled at 100 °C for 5min. The samples were then separated by 7.5% SDS–PAGE and the ubiquitinated substrates were detected by western blot analysis.
8
Analysis of Ubiquitinated Proteins by Ni-NTA Pulldown
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Ni-NTA Superflow Resin (Qaigen) was washed three times with Urea Buffer (10mM imidazole, 0.2% NP-40, 8 M urea, 500 mM NaH2PO4, 50 mM Tris HCl, pH 8.0). For each immunoprecipitation, 10 µl of resin was added per tube, and resuspended to 191 µl in Urea buffer. Extracts were supplemented with 100 µM of HIS6-Ubiquitin (Boston Biochem) and replication was carried out as described above. At the indicated time, 9 µl of extract was mixed with the bead mix and samples were incubated for one hour at room temperature, with end-over-end rotation. Resin was washed three times with urea buffer. All residual buffer was removed, and resin was boiled for 5 minutes in 30 μl sample buffer (125 mM Tris-HCl pH 6.8, 20% glycerol, 6.1% SDS, 0.01% bromophenol blue, 10% β-mercaptoethanol). 30 µl of 0.5 M imidazole was added to each sample and HIS6-tagged proteins were eluted off the resin for 60’ at room temperature, with gentle agitation. Resin was spun down at 1000 RCF for 1 minute, and the supernatant was removed. 10 µl of each sample was resolved on an SDS-PAGE gel alongside an input control and analyzed by Western blotting using the previously-described antibody against MCM749 (link). In extended Data Fig. 8D , a longer exposure of the IP lanes is shown, since they are far less intense than the input lanes.
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