Anti ubiquitin

Manufactured by R&D Systems

Sourced in United States

Anti-ubiquitin is a laboratory reagent used to detect and quantify the presence of ubiquitin, a small regulatory protein found in eukaryotic cells. It is commonly used in biochemical and cell biology research applications to study protein degradation and turnover pathways.

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Lab products found in correlation

Anti phospho p38, by R&D Systems (1 mentions) Mab3925, by R&D Systems (1 mentions) Anti gapdh, by R&D Systems (1 mentions) Anti p38, by R&D Systems (1 mentions) Anti nrf2, by R&D Systems (1 mentions) Hrp conjugated secondary antibody, by Cell Signaling Technology (1 mentions) Anti his, by Merck Group (1 mentions) Mutanbest kit, by Takara Bio (1 mentions) Ab126726, by Abcam (1 mentions) Anti flag, by Thermo Fisher Scientific (1 mentions) Anti ha, by Thermo Fisher Scientific (1 mentions) Anti α tubulin, by Merck Group (1 mentions) Anti h3, by Abcam (1 mentions) Nitrocellulose membrane, by Thermo Fisher Scientific (1 mentions) Anti mbp, by New England Biolabs (1 mentions) Stain free sds page gel, by Bio-Rad (1 mentions) Pmal c5x vector, by New England Biolabs (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions) Anti phospho threonine, by Merck Group (1 mentions) Anti human p53 do 1, by Santa Cruz Biotechnology (1 mentions)

5 protocols using anti ubiquitin

Western Blot Analysis of Cellular Proteins

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The relative amount of various proteins was determined by western blot analysis according to the protocol as described.²⁹ The antibodies used for immunoblot analysis were purchased from Cell Signaling Technologies (CST), Santa Cruz Biotechnology (SCBT) or R&D Biosystems (R&D): anti‐Ubiquitin (SCBT sc8017), anti‐Beclin‐1 (CST 3495), anti‐light chain (LC)‐3B (CST 2775), anti‐GAPDH (CST 2118), anti‐phospho‐p65 (CST 3033), anti‐p65 (CST 8242), anti‐phospho‐p38 (CST 9211), anti‐p38 (CST 9212), anti‐phospho‐AMPK (CST 2535), anti‐AMPK (CST 2532), anti‐TAK1 (CST 4505), anti‐Nrf2 (R&D Systems, MAB3925), and anti‐KEAP1 (CST 8647). HRP conjugated secondary antibodies were procured from Cell Signaling Technology. Band intensities were quantified with Image J software.

Roy A., Sharma A.K., Nellore K., Narkar V.A, & Kumar A. (2020). TAK1 preserves skeletal muscle mass and mitochondrial function through redox homeostasis. FASEB BioAdvances, 2(9), 538-553.

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Purification and Ubiquitination Assay of TaSDIR1-4A

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The ORF of TaSDIR1‐4A was introduced into the pCold‐TF‐QM‐Flag vector and expressed in Escherichia coli BL21(DE3). The expressed fusion protein His‐TaSDIR1‐4A‐Flag was purified according to the manufacturer's instructions (Sigma‐Aldrich). The conserved RING finger domain was mutated using a MutanBEST Kit (TaKaRa) according to the protocol provided by the manufacturer. The mutated primers used for Ala 230 to Ser 230 and for His 244 to Tyr 244 are shown in Table S1. The ubiquitination assays were performed as described previously (Park et al., 2018 (link)). The reaction system (30 μL final volume) comprised 1.5 μL 20× reaction buffer, 200 ng human E1 (UBE1), 200 ng human E2 (UBCh5b), 8 μg ubiquitin and 1 μg purified E3 (His‐TaSDIR1‐4A‐Flag). The reaction mixture was incubated at 30 °C for 2 h. Purified His‐TF‐Flag was used as a negative control. The products were separated by 8% sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and subjected to Western blot analysis using anti‐ubiquitin (R&D) and anti‐His (Sigma‐Aldrich) antibodies.

Meng Y., Lv Q., Li L., Wang B., Chen L., Yang W., Lei Y., Xie Y, & Li X. (2023). E3 ubiquitin ligase TaSDIR1‐4A activates membrane‐bound transcription factor TaWRKY29 to positively regulate drought resistance. Plant Biotechnology Journal, 22(4), 987-1000.

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Western Blot Analysis of Protein Targets

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After determining protein concentrations by the BCA assay, 5 or 10 μg of total protein per sample was used for LI-COR system-based Western blotting. After transfer, the nitrocellulose membrane was blocked in the Odyssey® blocking buffer. Primary and 800CW IRDye-conjugated secondary antibody incubations were also carried out in the Odyssey® blocking buffer. Primary antibodies used were anti-UPF1 (Abcam, ab86057), anti-α-tubulin (Sigma, T9026), anti-MYOD (Thermo, 5.8A, MA5-12902, for knockdown or overexpression experiments; Abcam, ab126726, for MB135-Tet-UPF1^{S124A/N138A/T139A} related experiments), anti-Ubiquitin (Boston Biochem, A-104), anti-FLAG (Thermo, FG4R, MA1-91878), anti-H3 (Abcam, ab1791) and anti-HA (Thermo, 2-2.2.14, 26183).

Feng Q., Jagannathan S, & Bradley R.K. (2017). The RNA surveillance factor UPF1 represses myogenesis via its E3 ubiquitin ligase activity. Molecular cell, 67(2), 239-251.e6.

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Ubiquitination Assay for Ptr Protein

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The entire coding region with 905 amino acids and partial coding sequence with 671 amino acids without predictive transmembrane domain of the N terminus of Ptr was cloned into SalI and EcoRI sites of the pMAL-c5X vector (NEB) to generate in-frame fusion proteins, respectively. The proteins were expressed at 20 °C and purified using amylose attached magnetic beads with an affinity for MBP fusion proteins according to the manufacturer’s protocol (NEB). In vivo ubiquitination was performed as described^{42 (link)}. To detect ubiquitination, the products were run through stain-free SDS-PAGE gels (Bio-Rad) and transferred onto Nitrocellulose membranes (Thermo Fisher Scientific, NC, USA). The membranes were blotted with anti-ubiquitin (Boston Biochem, MA, USA) and anti-MBP (NEB), respectively. The images were detected using ChemiDoc Imaging System (Bio-Rad, CA, USA). The primers and plasmid constructs for the ubiquitination assay study are listed in the Supplementary Tables 11 and 12, respectively.

Zhao H., Wang X., Jia Y., Minkenberg B., Wheatley M., Fan J., Jia M.H., Famoso A., Edwards J.D., Wamishe Y., Valent B., Wang G.L, & Yang Y. (2018). The rice blast resistance gene Ptr encodes an atypical protein required for broad-spectrum disease resistance. Nature Communications, 9, 2039.

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Western Blot Analyses using Multiple Antibodies

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Western blot analyses were performed as we described previously11 (link)17 (link)18 (link)20 (link)23 (link), using the following antibodies: anti-fortilin
(polyclonal antibody, MBL International, Woburn, MA, used for Fig. 1A,B, Fig. 2A, Fig.
3C, Fig. S2B, Fig. S2C, Fig.
S2D and Fig. S4B; monoclonal antibody [Clone 2C4], Abnova, Taiwan used
for Fig. 1F, Fig. 3A and Fig. S4F), anti-hemagglutinin (HA;
16B12, Bethyl Laboratory, Montgomery, TX), anti-FLAG (M2, Sigma), anti-human p53
(DO1, Santa Cruz), anti-PRX1 (goat polyclonal, PAB11441, Abnova, Taiwan),
anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH; 6C5, Fitzgerald),
anti-ubiquitin (BostonBiochem, Cambridge, MA), and anti-phosphothreonine
(Millipore, Billerica, MA) antibodies.

Chattopadhyay A., Pinkaew D., Doan H.Q., Jacob R.B., Verma S.K., Friedman H., Peterson A.C., Kuyumcu-Martinez M.N., McDougal O.M, & Fujise K. (2016). Fortilin potentiates the peroxidase activity of Peroxiredoxin-1 and protects against alcohol-induced liver damage in mice. Scientific Reports, 6, 18701.

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