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Ubiquitin enrichment kit

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The Ubiquitin Enrichment Kit is a tool designed for the isolation and enrichment of ubiquitinated proteins from cell lysates. It provides a simple and effective method to study ubiquitin-mediated cellular processes.

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Lab products found in correlation

Anti flag antibody, by Merck Group (1 mentions) Anti ha antibody, by Roche (1 mentions) α tubulin, by Santa Cruz Biotechnology (1 mentions) Vimentin, by Cell Signaling Technology (1 mentions) Anti ubiquitin antibody, by Thermo Fisher Scientific (1 mentions) Pierce ms compatible magnetic ip kit protein a g, by Thermo Fisher Scientific (1 mentions) Ubiquitin antibody p4d1, by Cell Signaling Technology (1 mentions)

5 protocols using ubiquitin enrichment kit

1

Detecting SNAIL Ubiquitination

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Whole cell lysates were prepared as described previously [17 (link)]. Primary antibodies used were SNAIL, COPS5, Vimentin from Cell Signaling Technology (Beverly, MA, USA), α-tubulin from Santa Cruz Biotechnology (Santa Cruz, CA, USA), anti-FLAG antibody (Sigma-Aldrich, St. Louis, MO, USA), and anti-HA antibody from Roche (Indianapolis, IN, USA). The ubiquitination of SNAIL was assessed by Ubiquitin Enrichment Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The immunoprecipitation lysates were subjected to Western blotting. The band intensities were measured by ImageJ and normalized to that of each control lane.
Watanabe K., Yokoyama S., Kaneto N., Hori T., Iwakami Y., Kato S., Hayakawa Y., Sakurai H., Fukuoka J, & Saiki I. (2018). COP9 signalosome subunit 5 regulates cancer metastasis by deubiquitinating SNAIL. Oncotarget, 9(29), 20670-20680.
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2

Ubiquitin Enrichment and Detection

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Ubiquitin Enrichment Kit obtained from Thermo Fisher Scientific (Rockford, IL) was used for detecting ubiquitinated proteins. Briefly, cells were treated with fenretinide in the presence or absence of various proteasome inhibitors such as MG132, and the cell lysates were (0.1 mg total protein) applied to a spin column containing 20 µl of polyubiquitin affinity resin. The spin column containing both sample and a positive control were incubated at 4°C overnight on an end-over-end rotator. After collecting the flow through by centrifuging the spin column at 5000 × g for 15 seconds, the column was washed three times with 300 µl of wash buffer. Then the proteins bound to the column was eluted by incubating the spin column at 70°C with 50 µl of SDS-PAGE sample loading buffer followed by centrifugation at 5000 × g for 30 seconds. The ubiquitin-enriched eluate was then analyzed by western blotting using mono- and polyubiquitin polyclonal antibody.
Samuel W., Kutty R.K., Duncan T., Vijayasarathy C., Kuo B.C., Chapa K.M, & Redmond T.M. (2014). Fenretinide induces ubiquitin-dependent proteasomal degradation of stearoyl-CoA desaturase in human retinal pigment epithelial cells. Journal of cellular physiology, 229(8), 1028-1038.
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3

PFKFB3 Polyubiquitination Regulation by HO-1/CO

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Endogenous ubiquitinated proteins in U937 cells were enriched by Ubiquitin Enrichment kit (Thermo Fisher Scientific, Waltham, MA) according to the manufacturer’s instructions. In brief, whole-cell extracts (1 mg) were incubated with polyubiquitin affinity resin for 4 h at 4 °C. Then, the resin was washed with TBS three times, followed by elution with 1 × sample buffer. The eluted ubiquitinated proteins were separated by SDS-PAGE, and then immunoblotting was performed with anti-PFKFB3 polyclonal antibody. To detect PFKFB3 polyubiquitination, HEK293 cells transfected with FLAG-tagged WT PFKFB3 (WT) or K142A mutant PFKFB3 were used. To examine the effects of HO-1/CO-mediated arginine methylation of PFKFB3 on polyubiqutination levels of the enzyme, the cells were treated with 25 μM haemin for 6 h or co-transfected with human PRMT1. The cell lysates (250 μg) were immunoprecipitated with anti-FLAG antibody for 4 h at 4 °C. The immunoprecipitates were analysed by SDS-PAGE, and followed by immunoblotting with anti-ubiquitin antibody (Thermo Fisher Scientific; 1,000x dilution). The whole-cell extracts were analysed with anti-FLAG, anti-HO-1 and anti-GAPDH antibodies as input.
Yamamoto T., Takano N., Ishiwata K., Ohmura M., Nagahata Y., Matsuura T., Kamata A., Sakamoto K., Nakanishi T., Kubo A., Hishiki T, & Suematsu M. (2014). Reduced methylation of PFKFB3 in cancer cells shunts glucose towards the pentose phosphate pathway. Nature Communications, 5, 3480.
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4

Enrichment and Identification of Polyubiquitinated Proteins in C. elegans

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Synchronized young adult animals were treated with Bortezomib (60 μM) for 8 h and worm pellets were washed 4 times and flash frozen in liquid nitrogen. Polyubiquitinated proteins were enriched using Ubiquitin Enrichment Kit (Thermo Scientific 89899) and eluted with 2× laemmli buffer. Lys48-specific anti-Ubiquitin rabbit clone Apu2 (Sigma 05-1207) was used for immunoprecipitation at 4 °C for 4 h. The immune-complex was captured using Pierce MS-Compatible Magnetic IP Kit Protein A/G (ThermoFisher 90409) and eluted with 2× laemmli buffer. Eluted proteins from both enrichment methods were resolved by SDS-PAGE and gel slices above 50 KDa were sent for protein ID using mass spectrometry. Ubiquitin antibody (P4D1, Cell Signaling Technology, 3936) was used for western blot to detect ubiquitinated proteins at 1:1000 dilution. Mass-spec and protein identification were performed by the UTSW proteomics core. For protein identification, samples were run on a Q-Exactive HF mass spectrometer coupled to an Ultimate 3000 RSLC-Nano liquid chromatography system. Data analysis was performed using Proteome Discoverer 3.0 SP1 using the C. elegans protein database from UniProt.
Yuan W., Weaver Y.M., Earnest S., Taylor CA I.V., Cobb M.H, & Weaver B.P. (2023). Modulating p38 MAPK signaling by proteostasis mechanisms supports tissue integrity during growth and aging. Nature Communications, 14, 4543.
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5

Ubiquitin Enrichment Quantification

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We measured ubiquitin enrichment by using the ubiquitin enrichment kit (ThermoFischer Scientist #1859662), according to the manufacturer's protocol.
Miotto B., Marchal C., Adelmant G., Guinot N., Xie P., Marto J.A., Zhang L, & Defossez P.A. (2018). Stabilization of the methyl-CpG binding protein ZBTB38 by the deubiquitinase USP9X limits the occurrence and toxicity of oxidative stress in human cells. Nucleic Acids Research, 46(9), 4392-4404.
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