AB309156

Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free

RabMAb
Recombinant
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Rabbit Recombinant Monoclonal UBC phospho S65 antibody. Carrier free. Suitable for WB, ICC/IF, Flow Cyt (Intra), I-ELISA and reacts with Human, Mouse, Synthetic peptide - Human samples.

View Alternative Names

Polyubiquitin-C, UBC

6 Images

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

This data was developed using ab309155, the same antibody clone in a different buffer formulation. Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized PC-3 (human prostate adenocarcinoma epithelial cell) cells labelling Ubiquitin (phospho S65) with ab309155 at 1/250 dilution (3.652 ug/ml), followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 dilution (2 ug/ml) (Green). Confocal image showing cytoplasmic and nuclear staining in PC-3 cells treated with CCCP (30μM) (ab141229) for 6 h. Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8). ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 dilution (2.5 ug/ml) (Red). The nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 dilution (2 ug/ml).

Flow Cytometry (Intracellular) - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

This data was developed using ab309155, the same antibody clone in a different buffer formulation. Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized PC-3 cells (human pancreas adenocarcinoma epithelial cells) treated with 30μM CCCP (ab141229) for 6 hours (Red) / untreated PC-3 cells (Green) cells labelling Ubiquitin (phospho S65) with ab309155 at 1/1000 dilution (0.1 ug) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody.

Indirect ELISA - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

I-ELISA

Supplier Data

Indirect ELISA - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

This data was developed using ab309155, the same antibody clone in a different buffer formulation. Indirect ELISA analysis of ab309155 at 0-1000 ng/ml. The secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) at 1/2500 dilution. Antigen : Human polyubiquitin-C (phospho S65) peptide1, Human polyubiquitin-C (phospho S65) peptide 2,Human polyubiquitin-C non-phospho peptide. Antigen concentration : 1000 ng/ml

Supplier Data

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

This data was developed using ab309155, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. In Western blot, Anti-Ubiquitin antibody (ab134953) staining at 1/1000 dilution. Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 97 seconds.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-ab309155'>ab309155</a>) at 1/1000 dilution

Lane 1:

Untreated Wild-type HEK-293T (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 2:

HEK-293T treated with 20 uM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 8 hours, whole cell lysate at 20 µg

Lane 3:

Untreated PINK1 knockout HEK-293T whole cell lysate at 20 µg

Lane 4:

PINK1 knockout HEK-293T treated with 20 uM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 8 hours, whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 8 kDa,200 kDa

false

Exposure time: 97s

Supplier Data

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

This data was developed using ab309155, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. The lysates were freshly made and used for Western Blotting immediately to minimize protein degradation. In Western blot, Anti-Ubiquitin antibody (ab134953) staining at 1/1000 dilution. Anti-GAPDH antibody (ab181602) loading control staining at 1/200000 dilution. Exposure time : 180 seconds.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-ab309155'>ab309155</a>) at 1/1000 dilution

Lane 1:

Untreated PC-3 (human prostate adenocarcinoma epithelial cell) whole cell lysate (untreated membrane) at 20 µg

Lane 2:

Untreated PC-3 whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 3:

PC-3 treated with 30 uM CCCP (<a href='/en-us/products/biochemicals/cccp-mitochondrial-oxidative-phosphorylation-uncoupler-ab141229'>ab141229</a>) for 2 hours, whole cell lysate (untreated membrane) at 20 µg

Lane 4:

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Observed band size: 8 kDa,200 kDa

false

Exposure time: 180s

Lab

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

This data was developed using ab309155, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

ab181602 was used as GAPDH loading control.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-ab309155'>ab309155</a>) at 1/1000 dilution

Lane 1:

Untreated MEF (mouse embryo fibroblast) whole cell lysate at 20 µg

Lane 2:

MEF treated with 5 μM Valinomycin for 24 hours whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 26 kDa

Observed band size: 8-200 kDa,27 kDa

false

Exposure time: 26s

Unconjugated

Anti-Ubiquitin (phospho S65) antibody [30H3/30K1]

Key facts

Host species

Rabbit

Clonality

Monoclonal

Clone number

30H3/30K1

Isotype

IgG

Carrier free

Yes

Reacts with

Human, Mouse

Applications

I-ELISA, Flow Cyt (Intra), WB, ICC/IF

applications

Immunogen

The exact immunogen used to generate this antibody is proprietary information.

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Reactivity data

{ "title": "Reactivity Data", "filters": { "stats": ["", "Species", "Dilution Info", "Notes"], "tabs": { "all-applications": {"fullname" : "All Applications", "shortname": "All Applications"}, "WB" : {"fullname" : "Western blot", "shortname":"WB"}, "ICCIF" : {"fullname" : "Immunocytochemistry/ Immunofluorescence", "shortname":"ICC/IF"}, "FlowCytIntra" : {"fullname" : "Flow Cytometry (Intracellular)", "shortname":"Flow Cyt (Intra)"}, "IELISA" : {"fullname" : "Indirect ELISA", "shortname":"I-ELISA"}, "Dot" : {"fullname" : "Dot Blot", "shortname":"Dot"} }, "product-promise": { "all": "all", "testedAndGuaranteed": "tested", "guaranteed": "expected", "predicted": "predicted", "notRecommended": "not-recommended" } }, "values": { "Human": { "WB-species-checked": "testedAndGuaranteed", "WB-species-dilution-info": "", "WB-species-notes": "", "ICCIF-species-checked": "testedAndGuaranteed", "ICCIF-species-dilution-info": "", "ICCIF-species-notes": "", "FlowCytIntra-species-checked": "testedAndGuaranteed", "FlowCytIntra-species-dilution-info": "", "FlowCytIntra-species-notes": "", "IELISA-species-checked": "guaranteed", "IELISA-species-dilution-info": "", "IELISA-species-notes": "", "Dot-species-checked": "notRecommended", "Dot-species-dilution-info": "", "Dot-species-notes": "" }, "Mouse": { "WB-species-checked": "testedAndGuaranteed", "WB-species-dilution-info": "", "WB-species-notes": "", "ICCIF-species-checked": "guaranteed", "ICCIF-species-dilution-info": "", "ICCIF-species-notes": "", "FlowCytIntra-species-checked": "guaranteed", "FlowCytIntra-species-dilution-info": "", "FlowCytIntra-species-notes": "", "IELISA-species-checked": "guaranteed", "IELISA-species-dilution-info": "", "IELISA-species-notes": "", "Dot-species-checked": "notRecommended", "Dot-species-dilution-info": "", "Dot-species-notes": "" }, "Synthetic peptide": { "WB-species-checked": "notRecommended", "WB-species-dilution-info": "", "WB-species-notes": "", "ICCIF-species-checked": "notRecommended", "ICCIF-species-dilution-info": "", "ICCIF-species-notes": "", "FlowCytIntra-species-checked": "notRecommended", "FlowCytIntra-species-dilution-info": "", "FlowCytIntra-species-notes": "", "IELISA-species-checked": "notRecommended", "IELISA-species-dilution-info": "", "IELISA-species-notes": "", "Dot-species-checked": "notRecommended", "Dot-species-dilution-info": "", "Dot-species-notes": "" }, "Synthetic peptide - Human": { "WB-species-checked": "notRecommended", "WB-species-dilution-info": "", "WB-species-notes": "", "ICCIF-species-checked": "notRecommended", "ICCIF-species-dilution-info": "", "ICCIF-species-notes": "", "FlowCytIntra-species-checked": "notRecommended", "FlowCytIntra-species-dilution-info": "", "FlowCytIntra-species-notes": "", "IELISA-species-checked": "testedAndGuaranteed", "IELISA-species-dilution-info": "", "IELISA-species-notes": "", "Dot-species-checked": "notRecommended", "Dot-species-dilution-info": "", "Dot-species-notes": "" } } }

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Product details

What are the advantages of a recombinant monoclonal antibody?
This product is a recombinant monoclonal antibody, which offers several advantages including:

- High batch-to-batch consistency and reproducibility
- Improved sensitivity and specificity
- Long-term security of supply
- Animal-free batch production

For more information, read more on recombinant antibodies.

Conjugation ready
Our carrier-free antibodies are typically supplied in a PBS-only formulation, purified and free of BSA, sodium azide and glycerol. This conjugation-ready format is designed for use with fluorochromes, metal isotopes, oligonucleotides, and enzymes, which makes them ideal for antibody labelling, functional and cell-based assays, flow-based assays (e.g. mass cytometry) and Multiplex Imaging applications.

Use our conjugation kits for antibody conjugates that are ready-to-use in as little as 20 minutes with 1 minute hands-on-time and 100% antibody recovery: available for fluorescent dyes, HRP, biotin and gold.

Compatibility
This product is compatible with the Maxpar^® Antibody Labeling Kit from Fluidigm, without the need for antibody preparation. Maxpar^® is a trademark of Fluidigm Canada Inc.

Want a custom formulation?
This antibody clone is manufactured by Abcam. If you require a custom buffer formulation or conjugation for your experiments, please contact orders@abcam.com

Collaborations
This antibody was developed with support from The Michael J. Fox Foundation.

Patented technology
Our RabMAb^® technology is a patented hybridoma-based technology for making rabbit monoclonal antibodies. For details on our patents, please refer to RabMAb^® patents.

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Properties and storage information

Form

Liquid

Purification technique

Affinity purification Protein A

Storage buffer

pH: 7.2 - 7.4 Constituents: PBS

Shipped at conditions

Blue Ice

Appropriate short-term storage conditions

+4°C

Appropriate long-term storage conditions

+4°C

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Supplementary information

This supplementary information is collated from multiple sources and compiled automatically.

Ubiquitin is a small regulatory protein found in almost all tissues of eukaryotic organisms. It has a molecular weight of approximately 8.6 kilodaltons. It functions mechanically by attaching to proteins through a process called ubiquitination which involves the formation of an isopeptide bond. Ubiquitin molecules can form polyubiquitin chains through different lysine residues such as K48 and K63 that determine their function. These chains label substrate proteins for various fates including degradation. Ubiquitin is expressed ubiquitously in cells reflecting its essential role in maintaining protein homeostasis.

Biological function summary

The ubiquitin system plays a critical role in regulating protein turnover and quality control within cells. It is part of a larger complex known as the ubiquitin-proteasome system (UPS) which is responsible for degrading proteins that need to be turned over. This process is essential for cell cycle control response to oxidative stress and DNA repair. Ubiquitin's role in tagging proteins for degradation or signaling allows cells to respond quickly to changes in their environment and maintain balance.

Pathways

Ubiquitin functions in several important biological pathways including the Wnt and NF-kB pathways. In the Wnt signaling pathway ubiquitination modulates the stability of key components thereby affecting the pathway's overall activity. In the NF-kB signaling pathway ubiquitin labels inhibitor proteins for degradation which releases and activates NF-kB. These pathways highlight ubiquitin's interaction with proteins such as beta-catenin in Wnt and IkB in NF-kB illustrating how it regulates diverse cellular processes.

The dysfunction of the ubiquitin system is linked to neurodegenerative diseases and cancers. Ubiquitin-related defects in protein degradation can lead to the buildup of unwanted proteins contributing to conditions like Parkinson's disease. Connections with cancer are evident as ubiquitin controls cell cycle proteins and aberrant ubiquitination may drive tumor growth and progression. The protein p53 known to be controlled by ubiquitination plays a significant role in cancer related mechanisms when dysregulated. Understanding and targeting ubiquitin-related pathways may provide new therapeutic opportunities for treating these conditions.

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Product protocols

For this product, it's our understanding that no specific protocols are required. You can visit:

Visit the General protocols
Visit the Troubleshooting

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Target data

Ubiquitin. Exists either covalently attached to another protein, or free (unanchored). When covalently bound, it is conjugated to target proteins via an isopeptide bond either as a monomer (monoubiquitin), a polymer linked via different Lys residues of the ubiquitin (polyubiquitin chains) or a linear polymer linked via the initiator Met of the ubiquitin (linear polyubiquitin chains). Polyubiquitin chains, when attached to a target protein, have different functions depending on the Lys residue of the ubiquitin that is linked : Lys-6-linked may be involved in DNA repair; Lys-11-linked is involved in ERAD (endoplasmic reticulum-associated degradation) and in cell-cycle regulation; Lys-29-linked is involved in proteotoxic stress response and cell cycle; Lys-33-linked is involved in kinase modification; Lys-48-linked is involved in protein degradation via the proteasome; Lys-63-linked is involved in endocytosis, DNA-damage responses as well as in signaling processes leading to activation of the transcription factor NF-kappa-B. Linear polymer chains formed via attachment by the initiator Met lead to cell signaling. Ubiquitin is usually conjugated to Lys residues of target proteins, however, in rare cases, conjugation to Cys or Ser residues has been observed. When polyubiquitin is free (unanchored-polyubiquitin), it also has distinct roles, such as in activation of protein kinases, and in signaling. During ubiquitination, the acceptor ubiquitin is positioned in the active site via direct interaction with the E2 ubiquitin-conjugating enzymes such as UBE2R2 (PubMed : 38326650). As a monoubiquitin, its C-terminal glycine is recognized as a C-degron by Cul2-RING (CRL2) E3 ubiquitin-protein ligase complexes (PubMed : 39548056).

See full target information Polyubiquitin-C phospho S65

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Product promise

We are committed to supporting your work with high-quality reagents, and we're here for you every step of the way. In the unlikely event that one of our products does not perform as expected, you're protected by our Product Promise.

For full details, please see our Terms & Conditions

Please note: All products are 'FOR RESEARCH USE ONLY. NOT FOR USE IN DIAGNOSTIC OR THERAPEUTIC PROCEDURES'.

For licensing inquiries, please contact partnerships@abcam.com

Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

Flow Cytometry (Intracellular) - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

Indirect ELISA - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - BSA and Azide free (AB309156)

Related conjugates and formulations (1)

Key facts

Host species

Clonality

Clone number

Isotype

Carrier free

Reacts with

Applications

Immunogen

Complementary Products

Secondary Antibodies

Primary Antibodies

Primary Antibodies

Primary Antibodies

Reactivity data

Product details

Properties and storage information

Form

Purification technique

Storage buffer

Shipped at conditions

Appropriate short-term storage conditions

Appropriate long-term storage conditions

Supplementary information

Biological function summary

Pathways

Product protocols

Target data

Product promise