For cell-free protein degradation assays, full-length MOC1 was amplified using primers (Supplementary Table 2 ) and cloned into the pMAL-c2x vector (NEW ENGLAND BioLabs, Catalog no. E8000S). Recombinant MBP-MOC1 proteins were expressed in E. coli BL21 (DE3) cells and purified through Amylose Resin (NEW ENGLAND BioLabs, Catalog no. E8021S). Seedlings of GFPOE, SLR1-GFPOE, sd1, GID1OE, and the corresponding WT were grown in a SANYO MLR Plant Growth Chamber (MLR-351) for 14 days at 28 °C during daytime and 25 °C at night with a 16:8 (day:night) photoperiod. The shoot bases (0–0.5 cm aboveground) were collected, and total proteins were extracted with degradation buffer (25 mM Tris-HCl, pH 7.5, 10 mM NaCl, 10 mM MgCl2, 4 mM PMSF, 5 mM dithiothreitol, and 10 mM ATP)42 (link) and adjusted to equal concentrations with the degradation buffer. Then 100 ng of purified MBP-MOC1 or His-trx-SLR1 protein was incubated in 100-μL extracts (containing 500 μg of total proteins) for the individual assays. The extracts were incubated at 28 °C, and samples were taken at the indicated intervals for protein blotting. Anti-MBP (NEW ENGLAND BioLabs, Catalog no. E8032), anti-His (Abmart, Catalog no. M20020), and anti-RPN6 (Enzo Life Sciences, Catalog no. BML-PW8370) antibodies were used at a 1:20,000 dilution.
Anti rpn6
Manufactured by Enzo Life Sciences
Anti-RPN6 is a laboratory reagent that targets the RPN6 protein. RPN6 is a critical component of the 26S proteasome, which is responsible for the degradation of proteins within the cell. Anti-RPN6 can be used to study the role of RPN6 in cellular processes.
Automatically generated - may contain errors
Lab products found in correlation
Amylose resin, by New England Biolabs (1 mentions)
Pmal c2x vector, by New England Biolabs (1 mentions)
Anti myc, by Abmart (1 mentions)
Chemidoc xr, by Bio-Rad (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
Mg132, by Merck Group (1 mentions)
Sds lysis buffer, by Beyotime (1 mentions)
Hrp conjugated goat anti rabbit igg, by Thermo Fisher Scientific (1 mentions)
Anti abi5, by Abcam (1 mentions)
Anti actin, by Abmart (1 mentions)
Supersignal west dura extended duration substrate, by Thermo Fisher Scientific (1 mentions)
Anti gfp, by Abcam (1 mentions)
5 protocols using anti rpn6
1
Cell-free Protein Degradation Assay
2
Quantifying Myc-KIX8 and Myc-KIX9 Levels
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3
Analyzing Arabidopsis Protein Stability
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Seeds of wild-type Col-0 and OX70-3, which were grown in the same growth chamber and harvested at the same day, were sown on MS medium containing 5 μM ABA and cultivated in a growth chamber under continuous white light for 3 d. After being washed twice with liquid MS medium, the seeds were transferred to fresh liquid MS medium supplemented with or without 50 μM MG132 (Sigma-Aldrich) or 100 μM CHX (Sigma-Aldrich), and harvested at the indicated time points. Subsequently, total proteins were extracted from the samples using SDS lysis buffer (Beyotime) and accurately quantified. After mixing with 5 × SDS-loading buffer, the proteins were boiled for 10 min, isolated on a 10% SDS-PAGE gel, and subsequently transferred to polyvinylidene fluoride (PVDF) membrane and incubated in blocking buffer (20mM Tris-HCl, 150 mM NaCl, 0.05% Tween 20, 5% nonfat milk powder) for 1 h at room temperature, then with 1:2000 anti-ABI5 (Abcam) or 1:1,000 anti-RPN6 (Enzo) for 12 h at 4°C, and followed with HRP-conjugated goat anti-rabbit IgG (Thermo) for 1 h. Finally, the PVDF membrane was overlaid on high-sig ECL western blotting substrate (Tanon) for 5 min and imaged using a CHEMI DOCTMXRS (Bio-rad).
4
Analyzing Ubiquitin Conjugate Levels in Arabidopsis
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To analyse the ubiquitin conjugate levels, total protein was extracted from 7-day-old Col (one group was treated with 10 μM MG132 for 2 h), ptre1 and PTRE1-overexpressing (p35S:PTRE1-GFP) seedlings, then subjected to SDS–PAGE and immunoblot analysis with antibodies. Antibodies used include anti-Ub (cat-sc-8017, 1:1,000, Santa); anti-RGA1 (cat-AS111630, 1:1,000, Agrisera); anti-PBA1 (cat- BML-PW0430, 1:1,000, Enzo); anti-RPN12A (cat-BML-PW0440, 1:1,000, Enzo); anti-RPN6 (cat-BML-PW8370, 1:1,000, Enzo); anti-ACTIN (cat-M20009, 1:5,000, Abmart); and anti-PTRE1 (1:1,000).
5
Protein Extraction and Western Blotting
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Total proteins from 100 mg of 4-day-old seedlings were extracted in 300 µl of extraction buffer (100 mM Tris-HCl pH 7.5, 100 mM NaCl, 5 mM EDTA pH 8.0, 5% SDS, 20% glycerol, 20 mM DTT, 40 mM β-mercaptoethanol, 2 mM PMSF, cOmpleteTM Protease Inhibitor Cocktail, 80 µM MG132 and 80 µM MG115, 1% phosphatase inhibitor cocktail 3, 10 mM N-ethylmaleimide, and 0.01% bromophenol blue). Samples were immediately boiled for 10 min and centrifuged at 16,000 × g for 10 min. Proteins were separated via SDS-PAGE and blotted onto a nitrocellulose membrane. The membrane was first probed with the indicated primary antibodies and then incubated with goat anti-rabbit (Bio-Rad, cat. no. 1706515) secondary antibodies conjugated with horseradish peroxidase at a 1:5000 dilution. The signals were detected via a chemiluminescence reaction using the SuperSignal West Dura Extended Duration Substrate (ThermoFisher Scientific, Waltham, MA). Rabbit polyclonal anti-PIF375 (link), goat polyclonal anti-HA (GenScript, cat. no. A00168), rabbit polyclonal anti-GFP (Abcam, cat. no. ab290), and rabbit polyclonal anti-RPN6 (Enzo Life Sciences, cat. no. BML-PW8370-0100) antibodies were used at a 1:1000 dilution.
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