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AB320097

Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free

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Rat Recombinant Monoclonal UBC phospho S65 antibody. Carrier free. Suitable for I-ELISA, WB, ICC/IF and reacts with Synthetic peptide - Human, Human, Mouse samples.

View Alternative Names

Polyubiquitin-C, UBC

5 Images
Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)
  • ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)

This data was developed using ab320096, 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 ab320096 at 1/50 (18.68 ug/ml) dilution, followed by ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) antibody at 1/1000 2ug/ml dilution (Green).

Confocal image showing increased staining in PC-3 cell line (shown in green). The counterstain was observed in magenta. Nuclear DNA was labelled with DAPI (shown in blue). Image was taken with a confocal microscope (Leica-Microsystems, TCS SP8).

ab195884 Anti-Tubulin rat monoclonal antibody - Microtubule Marker (Alexa Fluor® 647) was used to counterstain tubulin at 1/100 5ug/ml dilution (Magenta). The Nuclear counterstain was DAPI (Blue).

Secondary antibody only control : Secondary antibody is ab150157 Goat Anti-Rat IgG H&L (Alexa Fluor® 488) at 1/1000 2ug/ml dilution.

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)
  • WB

Supplier Data

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)

This data was developed using ab320096, the same antibody clone in a different buffer formulation.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</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:

PC-3 treated with 30 μM 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 3:

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

Lane 4:

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

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 180s

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)
  • WB

Supplier Data

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)

This data was developed using ab320096, the same antibody clone in a different buffer formulation.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

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

Lane 3:

Untreated PINK1 knockout HEK-293T whole cell lysate (untreated membrane) at 20 µg

Lane 4:

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

Lane 5:

Untreated Wild-type 293T (human embryonic kidney epithelial cell) whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 6:

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

Lane 7:

Untreated PINK1 knockout HEK-293T whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 8:

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

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 92s

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)
  • WB

Supplier Data

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)

This data was developed using ab320096, the same antibody clone in a different buffer formulation.

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

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/200000 dilution.

In Western blot, Anti-Ubiquitin antibody [EPR8830] (ab134953) staining at 1/1000 dilution.

All lanes:

Western blot - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) (<a href='/en-us/products/primary-antibodies/ubiquitin-phospho-s65-antibody-30h3-30k1-rat-igg2a-chimeric-ab320096'>ab320096</a>) at 1/1000 dilution

Lane 1:

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

Lane 2:

MEF treated with 5 μM Valinomycin for 24 hours whole cell lysate (untreated membrane) at 20 µg

Lane 3:

Untreated MEF (mouse embryo fibroblast) whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Lane 4:

MEF treated with 5 μM Valinomycin for 24 hours whole cell lysate (Alkaline phosphatase treated membrane) at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rat IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rat-igg-h-l-hrp-ab205720'>ab205720</a>) at 1/10000 dilution

Observed band size: 8-200 kDa,36 kDa

false

Exposure time: 92s

Indirect ELISA - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)
  • I-ELISA

Supplier Data

Indirect ELISA - Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric) - BSA and Azide free (AB320097)

This data was developed using ab320096, the same antibody clone in a different buffer formulation.

Indirect ELISA analysis of ab320096 at 1000-0 ng/ml. The Secondary antibody used was Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rat IgG (H+L) at 1 : 1000 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

  • Unconjugated

    Anti-Ubiquitin (phospho S65) antibody [30H3/30K1] - Rat IgG2a (Chimeric)

Key facts

Host species

Rat

Clonality

Monoclonal

Clone number

30H3/30K1

Isotype

IgG2a

Carrier free

Yes

Reacts with

Human, Mouse

Applications

WB, I-ELISA, 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"}, "IELISA" : {"fullname" : "Indirect ELISA", "shortname":"I-ELISA"}, "WB" : {"fullname" : "Western blot", "shortname":"WB"}, "ICCIF" : {"fullname" : "Immunocytochemistry/ Immunofluorescence", "shortname":"ICC/IF"}, "FlowCytIntra" : {"fullname" : "Flow Cytometry (Intracellular)", "shortname":"Flow Cyt (Intra)"} }, "product-promise": { "all": "all", "testedAndGuaranteed": "tested", "guaranteed": "expected", "predicted": "predicted", "notRecommended": "not-recommended" } }, "values": { "Human": { "IELISA-species-checked": "guaranteed", "IELISA-species-dilution-info": "", "IELISA-species-notes": "", "WB-species-checked": "testedAndGuaranteed", "WB-species-dilution-info": "", "WB-species-notes": "<p></p>", "ICCIF-species-checked": "testedAndGuaranteed", "ICCIF-species-dilution-info": "", "ICCIF-species-notes": "<p></p>", "FlowCytIntra-species-checked": "notRecommended", "FlowCytIntra-species-dilution-info": "", "FlowCytIntra-species-notes": "<p></p>" }, "Mouse": { "IELISA-species-checked": "guaranteed", "IELISA-species-dilution-info": "", "IELISA-species-notes": "", "WB-species-checked": "testedAndGuaranteed", "WB-species-dilution-info": "", "WB-species-notes": "<p></p>", "ICCIF-species-checked": "guaranteed", "ICCIF-species-dilution-info": "", "ICCIF-species-notes": "", "FlowCytIntra-species-checked": "notRecommended", "FlowCytIntra-species-dilution-info": "", "FlowCytIntra-species-notes": "" }, "Synthetic peptide - Human": { "IELISA-species-checked": "testedAndGuaranteed", "IELISA-species-dilution-info": "", "IELISA-species-notes": "<p></p>", "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": "" } } }
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Product details

ab320097 is the carrier-free version of ab320096.

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

This rat antibody has been engineered from a RabMAb parent antibody (ab309155). By necessity, some rabbit sequence is retained as part of the variable domain. When multiplexing with other rabbit-derived antibodies, using cross absorbed Fc-reactive secondary antibodies are recommended.

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.

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

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.

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

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Properties and storage information

Form
Liquid
Purification technique
Ion exchange chromatography
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:
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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).
See full target information Polyubiquitin-C phospho S65
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Product promise

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