Commercially available kits (TUBE1 and TUBE2, LifeSensors, Malvern, PA, USA) were utilized according to the manufacturer's protocol with slight modification. For the TUBE assay, A10 cells were treated with Pi and MG132 and the cell lysates were used for the assay. Either GST or GST-TUBE2 was loaded on Sepharose 4B beads and the cell lysates were applied and assayed as in the conventional GST pull-down method. The precipitates were then separated on an SDS–polyacrylamide gel electrophoresis gel and transferred to the polyvinylidene difluoride membrane. The membrane was blocked with 5% bovine serum albumin and probed with either anti-HDAC1 or anti-Flag antibody.
Tube2
Manufactured by LifeSensors
Sourced in United States
TUBE2 is a laboratory equipment designed for sample storage and preservation. It provides a controlled environment to maintain the integrity of biological samples during storage and transportation.
Automatically generated - may contain errors
Lab products found in correlation
Pr 619, by LifeSensors (2 mentions)
Tube1, by LifeSensors (1 mentions)
Mg132, by LifeSensors (1 mentions)
Protease inhibitor, by Roche (1 mentions)
Phosstop, by Roche (1 mentions)
O phenanthroline, by LifeSensors (1 mentions)
Halt protease and phosphatase inhibitor cocktail, by Thermo Fisher Scientific (1 mentions)
Fzr1 cdh1, by Thermo Fisher Scientific (1 mentions)
Conjugated secondary antibody, by Thermo Fisher Scientific (1 mentions)
Gapdh sc 32233, by Santa Cruz Biotechnology (1 mentions)
Protein 5x sample buffer, by Elpis Biotech (1 mentions)
Normal mouse igg, by Santa Cruz Biotechnology (1 mentions)
Protein a g plus agarose bead, by Santa Cruz Biotechnology (1 mentions)
Ubiquitin, by R&D Systems (1 mentions)
Anti ubiquitin, by Santa Cruz Biotechnology (1 mentions)
Anti ha, by Roche (1 mentions)
Anti flag, by Merck Group (1 mentions)
Adenosine triphosphate (atp), by Merck Group (1 mentions)
Complete protease inhibitor, by Roche (1 mentions)
6 protocols using tube2
1
TUBE-based HDAC1 Interaction Assay
2
Isolation and Analysis of Ubiquitinated Proteins
3
Isolation of polyubiquitinated proteins
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For polyubiquitinated protein isolation, we used agarose-coupled TUBE2 (LifeSensors). Immortalized B cells (4 × 106) and primary lymphocytes (1 × 107) were seeded and rested at least 1 h, and treated with PMA/ionomycin with or without z-VRPR-fmk for various times and then lysed in modified RIPA buffer supplemented with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific), and deubiquitinase inhibitors (5 mM O-phenanthroline and 50 μM PR619, LifeSensors). Cell lysates (550 μg) were mixed with 10 μl of pre-washed agarose-TUBE2 and incubated for 18 h at 4 °C. Agarose-TUBE2 was collected, washed and denatured in SDS–PAGE loading buffer. Samples were centrifuged and the supernatants were immunoblotted for total linear ubiquitin (LUB9 antibody, LifeSensors), NEMO (Santa Cruz Biotechnology; FL-419), RIP1 (Cell Signaling Technology; #4926) and K48-linked-polyubiquitin (Boston Biochem; #A-101). Scanned images of immunoblots were cropped in the final figures for clarity and conciseness. Full images of all blots are shown in Supplementary Fig. 8 .
4
Immunoprecipitation and Western Blot Analysis
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Mouse monoclonal antibodies against HA (sc-7392, 1:1000), Flag (Anti-DDDDK-tag, M185-3L, 1:1000) (MBL Life Science, Woburn, MA, USA), Histone H3 (CST-9751, 1:1000), GAPDH (sc-32233, 1:1000), ubiquitin (sc-8017, 1:1000) and normal mouse IgG (sc-2025, 1:1000) were purchased from Santa Cruz Biotech, Dallas, TX, USA; Histone H3 (CST-9751, 1:1000) were purchased from Cell Signaling Technology, Danvers, MA USA, rabbit polyclonal antibodies against FAH (LS-C165918, 1:1000) and FZR1/Cdh1 (Cat no. #34-2000, Invitrogen, 1:1000) were purchased from LS-Bio, Seattle, WA, USA and Invitrogen, Carlsbad, CA, USA, respectively. In addition, 488/594-conjugated secondary antibodies (Cat no. #A21207, Cat no. #A21203, 1:200) (Life Technologies, Carlsbad, CA, USA) were used. Protein A/G Plus Agarose beads (sc-2003, Santa Cruz Biotech, Dallas, TX, USA); protease inhibitor cocktail (Cat no. #B14012, Bimake.com, Houston, TX, USA), protein translation inhibitor cycloheximide (CHX; Cat no. #239765, Merck, Kenilworth, NJ, USA), RIPA buffer (Cat no. #R2002, Bioseong), Protein 5X sample buffer (Cat no. #EBA-1052, ELPIS BIOTECH, Taejon, Korea), proteasomal inhibitor MG132 (Cat no. #S2619, Selleckchem, Houston, TX, USA) and TUBE 2 (Cat no. #UM402, Life Sensors, Malvern, PA, USA) were also used.
5
In vitro and in vivo ubiquitination assays
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For in vitro ubiquitination assays, ubiquitination reactions were performed in a reaction mixture containing 50 mM Tris–HCl (pH 7.5), 20 mM MgCl2, 20 mM ATP (Sigma), 150 ng UBA1-His (E1), 250 ng UBC15-His (E2), 1 μg CUL3A-His (E3), 500 ng RBX1-GST, 250 ng LFH1-GST, 1 μg SVP-GFP-His, and 2 μg ubiquitin (Boston Biochem). The mixtures were incubated at 30°C for 3 h, and the reactions were terminated by adding SDS loading buffer. For in vivo ubiquitination assays, 7-day-old seedlings were treated with 150 μM MG132 and incubated for 12 h at 27°C. Frozen tissues were ground, and lysis buffer, which was recommended by the manufacturer of the precipitation resin (TUBE2; LifeSensors), was added to the homogenized powder. Cleared protein lysate was prepared by centrifugation (13 000 g, 10 min). Ten percent of the supernatant was retained to detect CUL3A or SVP in the input samples, and the remaining supernatant was incubated with TUBE2 resin at 4°C for 4 h or 6 h. For visualization, the precipitated proteins were separated by SDS–PAGE and immunoblotted with anti-ubiquitin (Santa Cruz, sc-9133), anti-Flag (Sigma-Aldrich, F1804), and anti-HA (Roche, 11867423001) antibodies.
6
Ubiquitin Enrichment from ES Cells
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E14Tg2a ES cells were lysed (50 mM Tris pH 7.5, 0.15 M NaCl, 1 mM EDTA, 1% NP-40, 10%
glycerol, 5 mM N-ethylmaleimide, Complete Protease Inhibitors (Roche)) on ice for 20
min. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C and soluble
supernatant incubated at 4°C overnight with TUBE2 or control agarose (LifeSensors)
prepared according to manufacturer’s instructions. Agarose beads were washed three
times in 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween and protein eluted with 2×
Laemmli SDS sample buffer. Data shown is representative of three replicates.
glycerol, 5 mM N-ethylmaleimide, Complete Protease Inhibitors (Roche)) on ice for 20
min. Cell lysates were centrifuged at 12,000 g for 10 min at 4°C and soluble
supernatant incubated at 4°C overnight with TUBE2 or control agarose (LifeSensors)
prepared according to manufacturer’s instructions. Agarose beads were washed three
times in 50 mM Tris pH 7.4, 150 mM NaCl, 0.1% Tween and protein eluted with 2×
Laemmli SDS sample buffer. Data shown is representative of three replicates.
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