Anti k63 ubiquitin

Manufactured by Merck Group

Sourced in United States

Anti-K63-ubiquitin is a laboratory reagent used to detect and quantify the presence of K63-linked polyubiquitin chains. It functions by specifically binding to K63-linked polyubiquitin chains, enabling researchers to study cellular processes involving this type of ubiquitination.

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Lab products found in correlation

Anti gapdh, by Abcam (6 mentions) Anti actin, by Cell Signaling Technology (5 mentions) Anti flag, by Merck Group (4 mentions) Pvdf membrane, by Thermo Fisher Scientific (3 mentions) Anti puromycin, by Merck Group (3 mentions) Anti ha, by Thermo Fisher Scientific (3 mentions) Anti ubiquitin k48, by Merck Group (2 mentions) Anti k48 ubiquitin, by Cell Signaling Technology (2 mentions) Anti dnp, by Merck Group (2 mentions) Anti k11 ubiquitin, by Merck Group (2 mentions) Anti k48, by Cell Signaling Technology (2 mentions) Anti myc, by Thermo Fisher Scientific (2 mentions) Anti mouse igg2a, by Abcam (2 mentions) Anti rad6, by Abcam (2 mentions) Anti rps3 us3, by Cell Signaling Technology (2 mentions) Anti rpl11 ul5, by Cell Signaling Technology (2 mentions) Anti rps20 us10, by Thermo Fisher Scientific (2 mentions) Anti ubc13, by Novus Biologicals (2 mentions) Anti ubiquitin, by Cell Signaling Technology (2 mentions) K48 ubiquitin, by Merck Group (1 mentions)

Spelling variants of this product

Ubiquitin (116 citations) Anti-ubiquitin (47 citations) Anti-K63 (6 citations) K63 Ubiquitin (4 citations)

12 protocols using anti k63 ubiquitin

Smad3 and Bcl-3 Protein Expression Analysis

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FLAG-tagged Bcl-3, or HA-tagged Smad3 were cloned into pcDNA3.1 and pcDNA3.0.
All PCR products were confirmed by sequencing. The following antibodies were
used: anti-Bcl-3, c-Myc, cyclinD1, p27, p21, vimentin, E-cadherin,
N-cadherin, ID1, FLAG, HA, ubiquitin (Santa Cruz Biotechnology, Inc., Santa
Cruz, CA, USA), anti-Snail, ID3, RBX1 (Proteintech), anti-phos-ERK,
phos-AKT, AKT, phos-Smad3, Smad3, phos-Smad2, Smad2, Lamin A/C (Cell
Signaling Technology, Boston, MA, USA), anti-GAPDH, donkey anti-Goat IgG
(HRP), goat anti-Mouse IgG (HRP), goat anti-Rabbit IgG (HRP) (KANGCHEN,
Shanghai, China), anti-Actin (Sigma-Aldrich, St. Louis, MO, USA),
anti-K63-ubiquitin, K48-ubiquitin (Millipore, Billerica, MA, USA), donkey
anti-goat coupled to AlexaFluor®488, and donkey anti-mouse or rabbit IgG
coupled to AlexaFluor680 (Invitrogen, Carlsbad, CA, USA). Anti-Smad3
Ser423/425 (Cell Signaling Technology), anti-Smad2/3 pT8,
anti-Smad2/3 pT179, anti-Smad3 pS204, anti-Smad3 pS208, anti-Smad3 pS213
were kind gifts from Dr Liu Fang.

Chen X., Cao X., Sun X., Lei R., Chen P., Zhao Y., Jiang Y., Yin J., Chen R., Ye D., Wang Q., Liu Z., Liu S., Cheng C., Mao J., Hou Y., Wang M., Siebenlist U., Eugene Chin Y., Wang Y., Cao L., Hu G, & Zhang X. (2016). Bcl-3 regulates TGFβ signaling by stabilizing Smad3 during breast cancer pulmonary metastasis. Cell Death & Disease, 7(12), e2508-.

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Antibody Immunoblotting for Cellular Signaling

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Antibodies against phosphorylated and total IRF3, IKKε, TBK1, NF-κB p65, ERK, JNK, and p38 were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti-TRAF3, anti-β-actin, and HRP-conjugated secondary antibodies were from Santa Cruz. Anti-Myc, anti-Flag, anti-HA, and anti-GFP were from Origene. Anti-PINK1, anti-Parkin, and anti-YAP1 were from Abcam Biotechnology (Cambridge, UK). Anti-K48-ubiquitin and anti-K63-ubiquitin were from Millipore (Kenilworth, USA).

Zhou J., Yang R., Zhang Z., Liu Q., Zhang Y., Wang Q, & Yuan H. (2019). Mitochondrial Protein PINK1 Positively Regulates RLR Signaling. Frontiers in Immunology, 10, 1069.

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Antibodies and Reagents for ARRDC4 and MDA5

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PMA were purchased from Sigma (St. Louis, MO, USA). Antibodies to ARRDC4 were from Abcam Inc. (ab209679) (Cambridge, MA, USA) or Santa Cruz Biotechnology Inc. (sc-135444) (Dallas, TX, USA). Antibodies to MDA5 (#21775-1-AP) was from Proteintech Group (Rosemont, IL, USA). Antibodies to MDA5 (D74E4, #5321), ERK (#9102), p-ERK (#9101), JNK (56G8, #9258), p-JNK (81E11, #4668), p38 (#9212), p-p38 (#9211), p65 (D14E12, #8242), p-p65 (93H1, #3033), IκBα (L35A5, #4814), IRF3 (D83B9, #4302) and p-IRF3 (#4947) were from Cell Signaling Technology (Beverly, MA, USA). Antibody to TRIM65 (HPA021578) was from Sigma, anti-EV71-VP1 antibody (ab169442) was from Abcam Inc. The antibodies to Flag-tag (#14793), Myc-tag (#2276), HA-tag (#3724) and V5-tag (D3H8Q, #13202) were from Cell Signaling Technology. The anti-K63-ubiquitin (#05-1313) was purchased from Millipore (San Diego, CA, USA).

Meng J., Yao Z., He Y., Zhang R., Zhang Y., Yao X., Yang H., Chen L., Zhang Z., Zhang H., Bao X., Hu G., Wu T, & Cheng J. (2017). ARRDC4 regulates enterovirus 71-induced innate immune response by promoting K63 polyubiquitination of MDA5 through TRIM65. Cell Death & Disease, 8(6), e2866-.

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CIITA Ubiquitination Study in COS Cells

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COS cells were plated at cell density of 8 × 10⁵/10 cm on tissue culture plates. Cells were transfected using GeneJuice (Merck Millipore, Darmstadt, Germany) as indicated with 5 μg of Myc-CIITA, Flag-pCAF, Flag-K141R, K144R, K141/144R CIITA, HA-K48 Ub, K63 Ub, HA-mono Ub, or pCDNA control. Twenty-four hours after transfections, cells were lysed in 1% NP40 buffer supplemented with EDTA-free protease inhibitors (Roche) on ice. Lysates were centrifuged, normalized for protein concentration, and precleared with Mouse IgG (Sigma-Aldrich) and Protein G (Thermo Fisher) followed by immunoprecipitation with either EZ view anti-c Myc affinity gel beads (Sigma-Aldrich) or with anti-Flag M2 affinity gel (Sigma-Aldrich). Immune complexes were denatured with Laemmli buffer, boiled, and separated by SDS-PAGE gel electrophoresis. Gels were transferred to nitrocellulose and were individually immunoblotted with anti-Myc (Abcam, Cambridge, MA), anti-Flag (Sigma-Aldrich), antiubiquitin (Life Sensors, Malvern, PA), anti-K48 ubiquitin (Cell Signaling, Danvers, MA), anti-K63 ubiquitin (Millipore), or with anti-GST (Abcam, Cambridge, MA). HRP conjugates were detected using HyGlo Chemiluminescent substrate (Denville). Protein normalization and equal loading were determined in lysates.

Morgan J.E, & Greer S.F. (2017). Pulling a Ligase out of a “HAT”: pCAF Mediates Ubiquitination of the Class II Transactivator. International Journal of Cell Biology, 2017, 8093813.

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Investigating AKT Regulation in Cellular Processes

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Chemicals were obtained from the following sources: cis-Diammineplatinum (II) dichloride (CDDP), N-acetylcysteine (NAC) and MG132 were purchased from Sigma-Aldrich (St. Louis, MO, USA). Antibodies were obtained from the following sources: anti-AKT (#9272 for western blot), anti-AKT (#2920 for proximity ligation assay, PLA), anti-p-AKT (Ser473), anti-Myc, anti-His, anti-FOXO1, anti-FOXO3, anti-FOXO4, anti-GAPDH, anti-Normal Rabbit IgG, horseradish peroxidase (HRP)-conjugated anti-mouse IgG and anti-rabbit IgG (Cell signaling Technology, Beverly, MA, USA), anti-K48 Ubiquitin, anti-K63 Ubiquitin, anti-α-Tubulin (Millipore, Temecula, CA, USA), anti-HA for western blot, anti-GFP (Santa Cruz Biotechnology, Santa Cruz, CA, USA), anti-MUL1, anti-Lamin A and anti-HA for ChIP assay (abcam, Cambridge, MA, USA), anti-hemagglutinin (HA), anti-GFP (Santa Cruz, Biotechnology, Santa Cruz, CA, USA), anti-Flag (Sigma-Aldrich), Alexa Flour 488-conjugated goat anti-Mouse and Alexa Flour 546-conjugated goat anti-Rabbit (Invitrogen, Carlsbad, CA, USA)

Kim S.Y., Kim H.J., Byeon H.K., Kim D.H, & Kim C.H. (2017). FOXO3 induces ubiquitylation of AKT through MUL1 regulation. Oncotarget, 8(66), 110474-110489.

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Investigating TRAF6 Ubiquitination in DT40 Cells

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WT DT40 cells and TAX1BP1^−/−/− cells were incubated for indicated time after CD40 activation and lysed in RIPA buffer. Lysates were incubated with anti-TRAF6 antibody (abcam) or relative non-immunized IgG for 3 h at 4 °C followed by protein G Sepharose for another 2 h. Proteins were detected by anti-TRAF6 (Santa Cruz Biotechnology), anti-β-actin (SIGMA) and anti-K63 Ubiquitin (Millipore).
Whole-cell lysates from DT40 cells were separated by SDS-PAGE gels and transferred to Immobilon-P membrane (Millipore). The membranes were blotted with the following antibodies: I-κBα, Phospho-I-κBα, JNK, Phospho-JNK, p44/42 MAPK, phospho-p44/42 MAPK (Cell Signaling), TAX1BP1 (Abnova), β-actin (SIGMA).

Matsushita N., Suzuki M., Ikebe E., Nagashima S., Inatome R., Asano K., Tanaka M., Matsushita M., Kondo E., Iha H, & Yanagi S. (2016). Regulation of B cell differentiation by the ubiquitin-binding protein TAX1BP1. Scientific Reports, 6, 31266.

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Yeast Cell Lysis and Western Blot

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For blot in Figure 3B and 5C–E, yeast cells grown to logarithmic phase (OD~0.5–0.6) were disrupted by glass-bead agitation at 4°C in buffer containing 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 20 mM iodoacetamide, 1X protease inhibitor cocktail set I (Sigma, #539131). Extracts were clarified by centrifugation, and protein concentration was determined by Bradford assay (Bio-Rad, #5000205) prior to western blotting. Proteins were separated by standard 10% or 12.5% SDS-PAGE loaded in Laemmli buffer and transferred to PVDF membrane (ThermoFisher, #88518). Immunoblotting was performed using the following antibodies: anti-K63 ubiquitin (EMD Millipore, #051308), anti-GAPDH (Abcam, #ab9485), anti-eiF2α-Phospho (Cell Signaling, #3398), anti-actin (Cell Signaling, #4967). For the blot in Figure S4E, yeast extracts were prepared from 25 mL of yeast cells grown to logarithmic phase (OD~0.5–0.6) by TCA precipitation. 10 μL samples were loaded on 4–20% Mini-Protean TGX gel (Bio-Rad, #4561096) and transferred to a PVDF membrane (Bio-Rad, #1704156). The proteins were detected using antibodies against eIF2α-Phospho (Abcam, #32157). The antibody against yeast eIF2α was kindly provided by the laboratory of Thomas Dever (NIH/NICHD).

Meydan S., Barros G.C., Simões V., Harley L., Cizubu B.K., Guydosh N.R, & Silva G.M. (2023). The ubiquitin conjugase Rad6 mediates ribosome pausing during oxidative stress. Cell reports, 42(11), 113359.

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Evaluating Ubiquitin Linkage Profiles

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Proteins were separated by standard 10% SDS-polyacrylamide gel electrophoresis (SDS–PAGE) loaded in Laemmli buffer containing 10 mM DTT. Samples were transferred to PVDF membrane and immunoblotting was performed using the following antibodies: anti-K63 ubiquitin (1:4,000 - cat# 05-1308, clone apu3, EMD Millipore), anti-K11 ubiquitin (1:1,000 - cat# MABS107, clone 2A3/2E6 EMD Millipore), anti-K48 (1:10,000 - cat# 4289, clone D9D5 - Cell Signaling), anti-actin (1:5,000 - cat# 4967 - Cell Signaling), anti-GAPDH (1:4,000 - cat# ab9485, Abcam), anti-DNP (1:8,000 - cat# D9656 - Sigma). Anti-mouse and anti-rabbit secondary antibodies conjugated with HRP and ECL prime detection reagents were acquired from GE Healthcare Life Sciences. All antibodies have been validated by the manufacturer or are expected to react with the species used in this study based on sequence similarity.

Silva G.M., Finley D, & Vogel C. (2015). K63 polyubiquitination is a new modulator of the oxidative stress response. Nature structural & molecular biology, 22(2), 116-123.

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Comprehensive Protein Immunoblotting Protocol

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Proteins were separated by standard 10% SDS-PAGE, with samples loaded in Laemmli buffer, transferred for 2h to a PVDF membrane, and immunoblotted using the following antibodies: anti-K63 ubiquitin (1:4,000; EMD Millipore, cat. No. 05–1308, clone apu3); anti-GAPDH (1:4,000; Abcam, cat. No. ab9485); anti-Rps10 (1:6,000, Sigma, cat no. WH0004736M1); anti-Rps6 (1:4,000; Abcam cat no. ab40820), anti-puromycin (1,2:500, EMD Millipore MABE343, clone 12D10), anti HA (1:2,500; Invitrogen, cat no. 71–5500), anti-K48 ubiquitin (1:8,000; Cell Signaling, cat. No 8081).

Back S., Gorman A.W., Vogel C, & Silva G.M. (2018). Site-specific K63 ubiquitinomics provides insights into translation regulation under stress. Journal of proteome research, 18(1), 309-318.

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Western Blot of Ubiquitin Modifications

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Proteins were separated by standard 10 - 15% SDS-PAGE loaded in Laemmli buffer. Samples were transferred to PVDF membrane (ThermoFisher), and immunoblotting was performed using the following antibodies: anti-K63 ubiquitin (1:4,000; EMD Millipore, cat. no. 05-1308, clone apu3), anti-actin (1:6,000; Cell Signaling, cat. no. 4967), anti-GAPDH (1:4,000; Abcam, cat. no. ab9485), anti-Myc (1:5,000; ThermoFisher, cat. no. R950-25 and PA1-981), anti-Rad6 (1:8,000; Abcam, cat. no. ab31917), anti-Rps3/uS3 (1:6,000; Cell Signaling, cat. no. 9538S), anti-Rpl11/uL5 (1:6,000; Cell Signaling, cat. no. 18163S), anti-HA (1:10,000; ThermoFisher, cat. no. 71-5500), anti-Rps20/uS10 (1:9,000; ThermoFisher, cat. no. PA5-75383), anti-ubiquitin (1:10,000; Cell Signaling Technology cat. No. 3936S), anti-FLAG (1:3,000; MilliporeSigma, cat. no. F3165), anti-puromycin (1:4,000; MilliporeSigma, cat. no. MABE343), anti-ubc13 (1:1,000; Novus Biologicals, cat. no. NBP1-76593). Anti-mouse IgG (1:8,000-12,000; Cytiva, ca. no. NA931), anti-rabbit IgG (1:6,000-12,000; Cytiva, cat. no. NA934), and Anti-mouse IgG2a (1:10,000-1:12,000; Abcam, cat. no. ab97245) secondary antibodies conjugated with HRP and ECL Prime detection reagents were acquired from Cytiva. All antibodies have been validated by the manufacturer or are expected to react with the species used in this study based on sequence similarity.

Simões V., Cizubu B.K., Harley L., Zhou Y., Pajak J., Snyder N.A., Bouvette J., Borgnia M.J., Arya G., Bartesaghi A, & Silva G.M. (2022). Redox-sensitive E2 Rad6 controls cellular response to oxidative stress via K63-linked ubiquitination of ribosomes. Cell reports, 39(8), 110860.

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