COS-7 cells were purchased from American Type Culture Collection (ATCC, Manassas, VA). Antibodies targeting SR-BI's C-terminal region (residues 450–509) or extracellular domain (residues 230–380) and anti-microtubule-associated protein 1A/1B-light chain 3B (LC3B) were obtained from Novus Biologicals, Inc. (Littleton, CO). Anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody was from Cell Signaling Technology (Danvers, MA). Anti-ubiquitin antibody was from Thermo Fisher Scientific (Waltham, MA). Peroxidase-conjugated anti-rabbit IgG and anti-mouse IgG were obtained from Amersham–GE Healthcare (Chicago, IL). Fluorescein isothiocyanate (FITC)-conjugated anti-rabbit IgG was purchased from BD Biosciences (Franklin Lakes, NJ). Human HDL was purchased from Alfa Aesar (Tewksbury, MA). [3H]-cholesterol and [125I]-sodium iodide were from PerkinElmer (Waltham, MA). [3H]-cholesteryl oleyl ether (COE) was from American Radiolabeled Chemicals, Inc. (St. Louis, MO). Chloroquine diphosphate salt, carbobenzoxy-Leu-Leu-leucinal (MG-132), cholesterol oxidase (Streptomyces), and thin-layer chromatography standards (cholesterol, 4-cholesten-3-one, and cholesteryl oleate) were obtained from Sigma–Aldrich (St. Louis, MO). All other reagents were of analytical grade.
Anti ubiquitin antibody
Manufactured by Thermo Fisher Scientific
Sourced in United States
The anti-ubiquitin antibody is a laboratory reagent used to detect and quantify the presence of ubiquitin, a small regulatory protein found in eukaryotic cells. This antibody can be used in various techniques, such as immunoblotting, immunoprecipitation, and immunohistochemistry, to study protein ubiquitination and its role in cellular processes.
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Lab products found in correlation
Cholesteryl oleate, by Merck Group (1 mentions)
Anti glyceraldehyde 3 phosphate dehydrogenase gapdh antibody, by Cell Signaling Technology (1 mentions)
Peroxidase conjugated anti rabbit igg, by Cytiva (1 mentions)
125i sodium iodide, by PerkinElmer (1 mentions)
3h cholesterol, by PerkinElmer (1 mentions)
Chloroquine diphosphate salt, by Merck Group (1 mentions)
Cos 7 cells, by American Type Culture Collection (1 mentions)
3h cholesteryl oleyl ether, by American Radiolabeled Chemicals (1 mentions)
Desmin antibody, by Abcam (1 mentions)
K48 linkage specific polyubiquitin antibody, by Cell Signaling Technology (1 mentions)
Pierce crosslink ip kit, by Thermo Fisher Scientific (1 mentions)
Recombinant human il 6, by R&D Systems (1 mentions)
Mg132, by R&D Systems (1 mentions)
Cycloheximide (chx), by Merck Group (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Thermo Fisher Scientific (1 mentions)
Ubiquitin enrichment kit, by Thermo Fisher Scientific (1 mentions)
Ch 2 microscope, by Olympus (1 mentions)
6 protocols using anti ubiquitin antibody
1
COS-7 Cells: Antibody and Radioactive Assays
2
Desmin Ubiquitination Analysis
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Desmin from the cultured cells was pulled down by desmin antibody (Abcam) using a Pierce Crosslink IP Kit (Thermo Scientific) following the manufacturer's instructions. The enriched desmin was then separated by 6% SDS-PAGE and immunoanalyzed by using an anti-ubiquitin antibody (Thermo Scientific), K48-linkage specific polyubiquitin antibody (Cell Signaling Technology) or K63-linkage specific polyubiquitin antibody (Cell Signaling Technology).
3
Ubiquitination Assay via Co-IP
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Ub IP for ubiquitination assay was carried by Co-Immunoprecipitation (Co-IP) Kit (Cat. #26149, Pierce). Cells treated with MG132 and transfected with siRNA were prepared in RIPA buffer. Then, the cell lysates were incubated with 10 μg anti-Ubiquitin antibody (1862775, Thermo Fisher Scientific) or normal mouse IgG and 50 μl Protein A + G beads at 4°C for 2 h with rotation. After the wash with cold PBS for 3 times, the ubiquitinated proteins were retrieved by eluting in 50 μl 2×SDS loading buffer and subjected to western blot.
4
Signaling Pathway Activation Assay
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Recombinant human IL-6 and MG132 were purchased from R&D Systems, Inc. (Minneapolis, MN, USA). CHX was purchased from Sigma Aldrich (St. Louis, MO, USA). Primary antibodies for p-STAT3Y705, STAT3, IKKα, IKKβ, IKKγ, p100, HA-tag (anti-rabbit), and His-tag (rabbit) were purchased from Cell Signaling Technology (Danvers, MA, USA). Primary antibody against His-tag (mouse) was purchased from Santa Cruz Biotech (Santa Cruz, CA, USA). An anti-ubiquitin antibody was obtained from Thermo Fisher Scientific (Waltham, MA, USA). DAPI was purchased from Invitrogen.
5
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.
6
Ubiquitin Staining in Autophagy-Deficient Cells
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