Ubiquitin Recombinant Rabbit Monoclonal Antibody [ST46-03]

Overview

Product Name

Ubiquitin Recombinant Rabbit Monoclonal Antibody [ST46-03] - BSA and Azide free

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Synthetic peptide within human Ubiquitin aa 1-50.

Species Reactivity

Human, Mouse, Rat

Validated Applications

WB, IF-Cell, IF-Tissue, IHC-P, FC

Positive Control

HeLa cell lysate, HeLa treated with 10μM MG-132 for 6 hours cell lysate, NIH/3T3 cell lysate, NIH/3T3 treated with 10μM MG-132 for 8 hours cell lysate, PC-12 cell lysate, 293T cell lysate, HepG2 cell lysate, HepG2, NIH/3T3 cells treated with 10µM MG-132 for 6 hours, PC-12 cells treated with 10µM MG-132 for 6 hours, human esophagus tissue, mouse esophagus tissue, rat esophagus tissue, mouse brain tissue, rat brain tissue.

Conjugation

unconjugated

Clone Number

ST46-03

Product Features

Form

Liquid

Concentration

1mg/ml

Storage Instructions

Store at 2-8℃. Avoid freeze.

Storage Buffer

1*PBS (pH7.4).

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

WB
1:5,000-1:10,000

IF-Cell
1:200-1:500

IF-Tissue
1:500-1:2,000

IHC-P
1:1,0000

FC
1:1,000

Target

Function

Ubiquitin is a conserved polypeptide unit that plays an important role in the ubiquitin-proteasome pathway. 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 lysosomal degradation; 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. This antibody reacts with ubiquitin, a polypeptide w/ Mr. of approx. 8.5kD. It reacts with physiologically occurring conjugates of ubiquitin and intracellular proteins. Specifically recognizes ubiquitinated cytoplasmic inclusion bodies.

Background References

1. Zhang P et al. Endometrial cancer-associated mutants of SPOP are defective in regulating estrogen receptor-a protein turnover. Cell Death Dis 6:e1687 (2015).

2. Ye X et al. Characterization of PHB1 and its role in mitochondrial maturation and yolk platelet degradation during development of Artemia embryos. PLoS One 9:e109152 (2014).

Sequence Similarity

Belongs to the ubiquitin family.

Post-translational Modification

[Ubiquitin]: Phosphorylated at Ser-65 by PINK1 during mitophagy. Phosphorylated ubiquitin specifically binds and activates parkin (PRKN), triggering mitophagy. Phosphorylation does not affect E1-mediated E2 charging of ubiquitin but affects discharging of E2 enzymes to form polyubiquitin chains. It also affects deubiquitination by deubiquitinase enzymes such as USP30.; [Ubiquitin]: Mono-ADP-ribosylated at the C-terminus by PARP9, a component of the PPAR9-DTX3L complex. ADP-ribosylation requires processing by E1 and E2 enzymes and prevents ubiquitin conjugation to substrates such as histones.

Subcellular Location

Cytoplasm, Nucleus, Membrane, Mitochondrion.

UNIPROT

SwissProt: P0CG47 Human

SwissProt: P0CG48 Human

SwissProt: P62979 Human

SwissProt: P62987 Human

SwissProt: P0CG49 Mouse

SwissProt: P62991 Mouse

SwissProt: P0CG51 Rat

SwissProt: Q63429 Rat

Synonyms

FLJ25987 antibody

MGC8385 antibody

Polyubiquitin B antibody

RPS 27A antibody

RPS27A antibody

UBA 52 antibody

UBA 80 antibody

UBA52 antibody

UBA80 antibody

UBB antibody

Expand

Images

☑ Cell treatment (CT)

Western blot analysis of Ubiquitin on different lysates with Rabbit anti-Ubiquitin antibody (HA750165) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution.

Lane 1: HeLa cell lysate
Lane 2: HeLa treated with 10μM MG-132 for 6 hours cell lysate
Lane 3: NIH/3T3 cell lysate
Lane 4: NIH/3T3 treated with 10μM MG-132 for 8 hours cell lysate
Lane 5: PC-12 cell lysate

Lysates/proteins at 20 µg/Lane.

Exposure time: 1 minute 34 seconds; ECL: K1801;

4-20% SDS-PAGE gel.

Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA750165) at 1/5,000 dilution and competitor's antibody at 1/2,000 dilution were used in 5% NFDM/TBST at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.
Immunocytochemistry analysis of HepG2 cells labeling Ubiquitin with Rabbit anti-Ubiquitin antibody (HA750165) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Ubiquitin antibody (HA750165) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
☑ Cell treatment (CT)

Immunocytochemistry analysis of NIH/3T3 cells treated with 10µM MG-132 for 6 hours labeling Ubiquitin with Rabbit anti-Ubiquitin antibody (HA750165) at 1/500 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Ubiquitin antibody (HA750165) at 1/500 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
☑ Cell treatment (CT)

Immunocytochemistry analysis of PC-12 cells treated with 10µM MG-132 for 6 hours labeling Ubiquitin with Rabbit anti-Ubiquitin antibody (HA750165) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 20 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 5 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Ubiquitin antibody (HA750165) at 1/200 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

Beta tubulin (M1305-2, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.
Immunohistochemical analysis of paraffin-embedded human esophagus tissue with Rabbit anti-Ubiquitin antibody (HA750165) at 1/10,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750165) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse esophagus tissue with Rabbit anti-Ubiquitin antibody (HA750165) at 1/10,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750165) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat esophagus tissue with Rabbit anti-Ubiquitin antibody (HA750165) at 1/10,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750165) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded mouse brain tissue with Rabbit anti-Ubiquitin antibody (HA750165) at 1/10,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750165) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Immunohistochemical analysis of paraffin-embedded rat brain tissue with Rabbit anti-Ubiquitin antibody (HA750165) at 1/10,000 dilution.

The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) (high pressure) for 2 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (HA750165) at 1/10,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.
Flow cytometric analysis of HepG2 cells labeling Ubiquitin.

Cells were fixed and permeabilized. Then stained with the primary antibody (HA750165, 1μg/mL) (red) compared with Rabbit IgG Isotype Control (green). After incubation of the primary antibody at +4℃ for an hour, the cells were stained with a iFluor™ 488 conjugate-Goat anti-Rabbit IgG Secondary antibody (HA1121) at 1/1,000 dilution for 30 minutes at +4℃. Unlabelled sample was used as a control (cells without incubation with primary antibody; black).