ET1609-21

Ubiquitin Recombinant Rabbit Monoclonal Antibody [ST46-03]

cat.: ET1609-21

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, FC
Clonality:	Monoclonal
Clone number:	ST46-03

Form:	Liquid
Storage condition:	Store at +4℃ after thawing. Aliquot store at -20℃ or -80℃. Avoid repeated freeze / thaw cycles.
Storage buffer:	1*TBS (pH7.4), 0.05% BSA, 40% Glycerol. Preservative: 0.05% Sodium Azide.
Concentration:	1ug/ul
Purification:	Protein A affinity purified.
Isotype:	IgG
Immunogen:	Synthetic peptide within human Ubiquitin aa 1-50.
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, HepG2, mouse testis tissue, mouse placenta tissue, mouse brain tissue, human esophagus tissue, 293T cell lysate, HepG2 cell lysate.
Subcellular location:	Cytoplasm, Nucleus, Membrane, Mitochondrion.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P FC	1:1,000-1:5,000 1:1:500 1:50-1:200 1:200-1:500 1:1,000
Uniprot #:	SwissProt: P0CG47 Human \| P0CG48 Human \| P62979 Human \| P62987 Human \| P0CG49 Mouse \| P62991 Mouse \| P0CG51 Rat \| Q63429 Rat
Alternative names:	FLJ25987 MGC8385 Polyubiquitin B RPS 27A RPS27A UBA 52 UBA 80 UBA52 UBA80 UBB UBB_HUMAN UBC UBCEP 1 UBCEP 2 UBCEP1 UBCEP2 Ubiquitin Ubiquitin B

Images

Fig1: Western blot analysis of Ubiquitin on different lysates with Rabbit anti-Ubiquitin antibody (ET1609-21) 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;

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 (ET1609-21) 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.

Fig2: Immunocytochemistry analysis of HepG2 cells labeling Ubiquitin with Rabbit anti-Ubiquitin antibody (ET1609-21) 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 (ET1609-21) 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.

	Fig3: Western blot analysis of Ubiquitin on different lysates with Rabbit anti-Ubiquitin antibody (ET1609-21) at 1/1,000 dilution. Lane 1: PC-12 cell lysate Lane 2: HeLa cell lysate Lane 3: NIH/3T3 cell lysate Lane 4: 293T cell lysate Lane 5: HepG2 cell lysate Lysates/proteins at 20 µg/Lane. Exposure time: 2 minutes; 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 (ET1609-21) at 1/1,000 dilution was used in 5% NFDM/TBST at room temperature for 2 hours. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/100,000 dilution was used for 1 hour at room temperature
	Fig4: Immunohistochemical analysis of paraffin-embedded mouse testis tissue using anti-Ubiquitin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-21, 1/50) for 30 minutes 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.
	Fig5: Immunohistochemical analysis of paraffin-embedded mouse brain tissue using anti-Ubiquitin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-21, 1/50) for 30 minutes 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.

	Fig6: Immunohistochemical analysis of paraffin-embedded mouse placenta tissue using anti-Ubiquitin antibody. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 8.0-8.4) for 20 minutes.The tissues were blocked in 5% BSA for 30 minutes at room temperature, washed with ddH₂O and PBS, and then probed with the primary antibody (ET1609-21, 1/50) for 30 minutes 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.
	Fig7: Immunohistochemical analysis of paraffin-embedded human esophagus tissue with Rabbit anti-Ubiquitin antibody (ET1609-21) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) 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 (ET1609-21) at 1/200 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.
	Fig8: Flow cytometric analysis of HepG2 cells labeling Ubiquitin. Cells were fixed and permeabilized. Then stained with the primary antibody (ET1609-21, 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).

Note: All products are “FOR RESEARCH USE ONLY AND ARE NOT INTENDED FOR DIAGNOSTIC OR THERAPEUTIC USE”.