ET1703-22

PGP9.5 Recombinant Rabbit Monoclonal Antibody [JM10-59]

cat.: ET1703-22

Product Type:	Recombinant Rabbit monoclonal IgG, primary antibodies
Species reactivity:	Human, Mouse, Rat
Applications:	WB, IF-Cell, IF-Tissue, IHC-P, IP, FC, IHC-Fr
Clonality:	Monoclonal
Clone number:	JM10-59

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: 25 kDa
Isotype:	IgG
Immunogen:	Synthetic peptide within Human PGP95 aa 191-223 / 223.
Positive control:	A-172 cell lysate, SHG-44 cell lysate, U-87 MG cell lysate, SH-SY5Y cell lysate, NCI-H1299 cell lysate, A549 cell lysate, 293T cell lysate, Neuro-2a cell lysate, C6 cell lysate, PC-12 cell lysate, mouse brain tissue lysate, rat brain tissue lysate, N2A, PC-12, mouse spinal cord tissue, mouse skin tissue, mouse brain tissue, mouse colon tissue, mouse cerebrum tissue.
Subcellular location:	Cytoplasm, Endoplasmic reticulum membrane.
Recommended Dilutions: WB IF-Cell IF-Tissue IHC-P IP FC IHC-Fr	1:2,000-1:5,000 1:100-1:500 1:100-1:1,000 1:1,000-1:5,000 Use at an assay dependent concentration. 1:1,000 1:200-1:500
Uniprot #:	SwissProt: P09936 Human \| Q9R0P9 Mouse \| Q00981 Rat
Alternative names:	Epididymis luminal protein 117 Epididymis secretory protein Li 53 HEL 117 HEL S 53 NDGOA Neuron cytoplasmic protein 9.5 OTTHUMP00000218137 OTTHUMP00000218139 OTTHUMP00000218140 OTTHUMP00000218141 Park 5 PARK5 PGP 9.5 PGP9.5 PGP95 Protein gene product 9.5 Ubiquitin C terminal esterase L1 Ubiquitin C terminal hydrolase Ubiquitin C terminal hydrolase L1 Ubiquitin carboxyl terminal esterase L1 Ubiquitin carboxyl terminal hydrolase isozyme L1 Ubiquitin carboxyl-terminal hydrolase isozyme L1 Ubiquitin thioesterase L1 Ubiquitin thiolesterase Ubiquitin thiolesterase L1 UCH-L1 UCHL1 UCHL1_HUMAN

Images

Fig1: Western blot analysis of PGP9.5 on different lysates with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/2,000 dilution.

Lane 1: A-172 cell lysate (20 µg/Lane)
Lane 2: SHG-44 cell lysate (20 µg/Lane)
Lane 3: U-87 MG cell lysate (20 µg/Lane)
Lane 4: SH-SY5Y cell lysate (20 µg/Lane)
Lane 5: NCI-H1299 cell lysate (20 µg/Lane)
Lane 6: A549 cell lysate (20 µg/Lane)
Lane 7: 293T cell lysate (20 µg/Lane)
Lane 8: Neuro-2a cell lysate (20 µg/Lane)
Lane 9: C6 cell lysate (20 µg/Lane)
Lane 10: PC-12 cell lysate (20 µg/Lane)
Lane 11: Mouse brain tissue lysate (30 µg/Lane)
Lane 12: Rat brain tissue lysate (30 µg/Lane)
Lane 13: LNCaP cell lysate (negative) (20 µg/Lane)
Lane 14: K-562 cell lysate (negative) (20 µg/Lane)

Predicted band size: 25 kDa
Observed band size: 25 kDa

Exposure time: 7 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 (ET1703-22) at 1/2,000 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: Western blot analysis of PGP9.5 on different lysates with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/5,000 dilution.

Lane 1: A549-si NT cell lysate (10 µg/Lane)
Lane 2: A549-si PGP9.5 cell lysate (10 µg/Lane)

Predicted band size: 25 kDa
Observed band size: 25 kDa

Exposure time: 3 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 (ET1703-22) at 1/5,000 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.

	Fig3: Immunohistochemical analysis of paraffin-embedded human appendix tissue with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/5,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1703-22) at 1/5,000 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.
	Fig4: Immunohistochemical analysis of paraffin-embedded human pancreas tissue with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/5,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1703-22) at 1/5,000 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.
	Fig5: Immunohistochemical analysis of paraffin-embedded mouse colon tissue with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/5,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1703-22) at 1/5,000 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.

	Fig6: Immunohistochemical analysis of paraffin-embedded rat pancreas tissue with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/5,000 dilution. The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 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 (ET1703-22) at 1/5,000 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.
	Fig7: Immunocytochemistry analysis of SH-SY5Y(+)/LNCAP(-) cells labeling PGP9.5 with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/250 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-PGP9.5 antibody (ET1703-22) at 1/250 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.
	Fig8: Immunocytochemistry analysis of Neuro-2a cells labeling PGP9.5 with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/100 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-PGP9.5 antibody (ET1703-22) at 1/100 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.

	Fig9: Immunocytochemistry analysis of PC-12 cells labeling PGP9.5 with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/100 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-PGP9.5 antibody (ET1703-22) at 1/100 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.
	Fig10: Immunofluorescence analysis of frozen mouse cerebrum tissue with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/500 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1703-22, green) at 1/500 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).
	Fig11: Immunofluorescence analysis of frozen mouse pancreas tissue with Rabbit anti-PGP9.5 antibody (ET1703-22) at 1/200 dilution. The section was pre-treated using heat mediated antigen retrieval with sodium citrate buffer (pH 6.0) for about 2 minutes in microwave oven. The tissues were blocked in 10% negative goat serum for 1 hour at room temperature, washed with PBS, and then probed with the primary antibody (ET1703-22, green) at 1/200 dilution overnight at 4 ℃, washed with PBS. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. Nuclei were counterstained with DAPI (blue).

Fig12: Flow cytometric analysis of SH-SY5Y cells labeling PGP9.5.

Cells were fixed and permeabilized. Then stained with the primary antibody (ET1703-22, 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).

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