ET1612-82

UBA1 Recombinant Rabbit Monoclonal Antibody [SD08-62]

cat.: ET1612-82

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:	SD08-62

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.
Molecular weight:	Predicted band size: 118 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human UBA1 aa 1,009-1,058 / 1,058.
Positive control:	K-562 cell lysate, HeLa cell lysate, U-87 MG cell lysate, PC-12 cell lysate, Mouse spleen tissue lysate, Rat spleen tissue lysate, HeLa, NIH/3T3, PC-12, human colon cancer tissue, human kidney tissue, mouse kidney tissue, rat kidney tissue.
Subcellular location:	Nucleus, Mitochondrion, Cytoplasm.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P FC	1:1,000-1:2,000 1:200 1:50-1:200 1:200-1:1,000 1:1,000
Uniprot #:	SwissProt: P22314 Human \| Q02053 Mouse \| Q5U300 Rat
Alternative names:	A1S9 A1S9 protein A1S9T and BN75 temperature sensitivity complementing A1S9T A1ST AMCX1 CFAP124 CTD-2522E6.1 GXP 1 GXP1 MGC4781 POC20 POC20 centriolar protein homolog Protein A1S9 SMAX2 Uba1 UBA1, ubiquitin-activating enzyme E1 homolog A UBA1_HUMAN UBA1A UBE 1 UBE 1X UBE1 UBE1X Ubiquitin activating enzyme E1 Ubiquitin-activating enzyme E1 Ubiquitin-like modifier-activating enzyme 1

Images

Fig1: Western blot analysis of UBA1 on different lysates with Rabbit anti-UBA1 antibody (ET1612-82) at 1/1,000 dilution.

Lane 1: K-562 cell lysate
Lane 2: HeLa cell lysate
Lane 3: U-87 MG cell lysate
Lane 4: PC-12 cell lysate
Lane 5: Mouse spleen tissue lysate
Lane 6: Rat spleen tissue lysate

Lysates/proteins at 20 µg/Lane.

Predicted band size: 118 kDa
Observed band size: 118 kDa

Exposure time: 25 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 (ET1612-82) at 1/1,000 dilution was 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 HeLa cells labeling UBA1 with Rabbit anti-UBA1 antibody (ET1612-82) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-UBA1 antibody (ET1612-82) 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 (HA601187, 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: Immunocytochemistry analysis of NIH/3T3 cells labeling UBA1 with Rabbit anti-UBA1 antibody (ET1612-82) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-UBA1 antibody (ET1612-82) 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 (HA601187, 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.

Fig4: Immunocytochemistry analysis of PC-12 cells labeling UBA1 with Rabbit anti-UBA1 antibody (ET1612-82) at 1/200 dilution.

Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 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-UBA1 antibody (ET1612-82) 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 (HA601187, 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.

	Fig5: Immunohistochemical analysis of paraffin-embedded human colon cancer tissue with Rabbit anti-UBA1 antibody (ET1612-82) 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 (ET1612-82) 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.
	Fig6: Immunohistochemical analysis of paraffin-embedded human kidney tissue with Rabbit anti-UBA1 antibody (ET1612-82) 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 (ET1612-82) 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.
	Fig7: Immunohistochemical analysis of paraffin-embedded mouse kidney tissue with Rabbit anti-UBA1 antibody (ET1612-82) at 1/1,000 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 (ET1612-82) at 1/1,000 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.

Fig8: Immunohistochemical analysis of paraffin-embedded rat kidney tissue with Rabbit anti-UBA1 antibody (ET1612-82) at 1/1,000 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 (ET1612-82) at 1/1,000 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.

Fig9: Flow cytometric analysis of HeLa cells labeling UBA1.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1612-82, 1/1,000) (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).

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