Protein Gene Product 9.5 antibody | 31A3
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 (7863-1004)used for the evaluation of PGP9.5 expression in rat brain by western blotting.
Image caption:
UCH-L1 exists in both cytosolic and membrane-associated forms in neurons. Cell lines contain much lower levels of membrane-associated UCH-L1 than neurons. A, UCH-L1 exists in cytosolic and membrane-enriched fractions of whole rat brain. Adult male rat brain was homogenized and subjected to subcellular fractionation. Fractions were immunoblotted for protein markers to verify enrichment. 30 μg of protein was loaded in each lane. Numbers shown are in kilodaltons relative to molecular weight standards. P1, nuclear-enriched fraction; P2, crude membranes and synaptosomes; P3, microsomes, including the endoplasmic reticulum and Golgi; S2, cytosol and microsomes; S3, cytosol; Mito, mitochondria; Syn, synaptosomes. B, UCH-L1 exists in cytosolic and membrane fractions of cultured cortical neurons. Rat cortical neuronal lysate was subjected to high-speed ultracentrifugation to isolate the soluble cytosolic supernatant and membrane pellet fractions. Protein distribution was tested by immunoblotting for soluble cytosolic protein (RhoGDI) and transmembrane protein (GluA1). A proportion of Rac1 in neurons is peripherally associated with the membrane via geranylgeranylation, and the endogenous distribution is preserved using this protocol. 15 μg of protein was loaded in each lane. C, relative proportions of UCH-L1 detected per microgram of protein in the cytosolic and membrane fractions of cultured cortical neurons. The UCH-L1 signal was quantified using Li-Cor Odyssey imaging software. a.u., arbitrary units. D, cell lines contain much lower levels of UCH-L1M than cultured cortical neurons. Lysates from the indicated clonal cell lines were subjected to high-speed ultracentrifugation to isolate the cytosolic and membrane fractions. Protein distribution was tested by immunoblotting for cytosolic protein (RhoGDI), and prenylated protein (Rac1). The distribution of Rac1 is preserved in cell lines, although the proportions vary between cell lines. UCH-L1 was not detected in the membrane fraction of COS7 or HEK293T cells and only to a minor extent with increased gain in N2a cells. 20 μg of protein was loaded in each lane. E, relative proportions of UCH-L1 detected per microgram of protein in the cytosolic and membrane fractions of the indicated cell lines. The UCH-L1 signal was quantified using Li-Cor Odyssey imaging software. F, serum starvation induces outgrowth of neuronal-like processes in N2a cells. GFP-transfected N2a cells were exposed to serum starvation for 24 h and fixed for confocal microscopy. Cell outlines were identified, and images were prepared using ImageJ software with MacBiophotonics plug-ins. G, differentiation does not alter the proportion of UCH-L1M in N2a cells. Outgrowth of neuronal-like processes was induced in N2a cells with 24-h serum starvation, and then cells were subjected to ultracentrifugation as before. The proportion of UCH-L1M remained unchanged compared with untreated N2a cells. H, relative proportions of UCH-L1M detected per microgram of protein in the membrane fraction of N2a cells. The UCH-L1 signal was quantified using Li-Cor Odyssey imaging software.
From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM.
The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 used for the evaluation of PGP9.5 expression in rat brain by western blotting.
Image caption:
The farnesyltransferase inhibitor FTI-276 does not reduce UCH-L1M in neurons. A, membrane-associated H-ras, but not UCH-L1, is decreased by farnesyltransferase inhibition. Cultured cortical neurons were treated with a DMSO control or increasing amounts of the farnesyltransferase inhibitor FTI-276 (Calbiochem), as indicated, for 48 h and then lysed and subjected to high-speed ultracentrifugation to separate the cytosolic and membrane fractions. Immunoblotting for the known farnesylation substrate H-ras shows an increasing shift from a membrane-associated form to a cytosolic form as the concentration of FTI-276 increases, whereas the proportion of UCH-L1 remains unaffected. B, relative proportions of UCH-L1M and H-rasM detected per microgram of protein in the FTI-treated membrane fractions relative to the DMSO control (n = 6 for the DMSO control and 500 nm, and n = 5 for both 1 and 3 μm). Data were analyzed using one-way analysis of variance with post hoc Bonferroni test. *, p <0.05; **, p <0.001. a.u., arbitrary units.
From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM. The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 (7863-1004) used for the evaluation of PGP9.5 expression in rat brain by western blotting.
Image caption:
Selected mutations do not reduce membrane-associated UCH-L1M. A, three-dimensional models showing the location of visible mutated or deleted residues in UCH-L1 using Cn3D software with UCH-L1 PDB codes 2ETL and 3KW5 (bound to UbVME). The Ile-93 residue is buried internally. B, effect of individual UCH-L1 cysteine mutations on UbVME binding in N2a cells. N2a cells were transfected with HA-UCH-L1 Cys-to-Ser point mutants as indicated. Cell lysates were incubated with 2 μm UbVME to assess the effect of mutation on substrate binding and then immunoblotted (IB) to detect infected HA-UCH-L1 constructs and total (endogenous + infected) UCH-L1. C, 3CS (C90S, C152S, and C220S) and D30K mutations abolish UbVME binding, but I93M does not. Cultured cortical neurons were infected with Sindbis virus expressing the indicated HA-tagged UCH-L1 constructs. Lysates were incubated with 2 μm UbVME to assess the effect of mutations on substrate binding and then immunoblotted to detect infected HA-UCH-L1 constructs and total (endogenous and infected) UCH-L1. D, 3CS (C90S, C152S, and C220S), D30K, and I93M mutations do not reduce the proportion of UCH-L1M. Cultured cortical neurons were infected with Sindbis virus expressing the indicated HA-tagged UCH-L1. Lysates were subjected to ultracentrifugation to separate cytosolic and membrane-associated proteins. The fractions were immunoblotted to detect infected HA-UCH-L1 constructs and total (endogenous and infected) UCH-L1. E, relative proportions of the indicated HA-UCH-L1M constructs detected per microgram of protein in the membrane fraction relative to the cytosolic fraction (WT, n=9; 3CS, n=3; D30K, n=5; I93M, n=3). a.u., arbitrary units.
From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM. The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 (7863-1004) used for the evaluation of PGP9.5 expression in rat brain by immunofluorescence.
Image caption:
Deletion of the final four residues of UCH-L1 leads to increased cell death. A, deletion of the final four residues leads to altered UCH-L1 distribution. Hippocampal neurons were infected with Sindbis virus expressing the indicated HA-UCH-L1 constructs to assess the distribution of total (endogenous and infected) UCH-L1 and infected HA-UCH-L1. HA-UCH-L1 distribution in WT, N-terminal truncation, and CTTΔ2-infected cells is comparable with endogenous UCH-L1. Infected protein is largely absent from the processes. B, deletion of the final four residues leads to increased cell death. The cell death assay measured the release of lactate dehydrogenase. Infection with Sindbis virus expressing the HA-CTTΔ4 construct caused a significant increase in cell death in cortical neurons compared with HA-WT. Control, uninfected. Data were analyzed using one-way analysis of variance with post hoc Bonferroni test. *, p <0.05. Control, HA-WT, and HA-CTTΔ4, n=5; HA-NTT, n=4; HA-CTTΔ2, n=3.
From: Bishop P, Rubin P, Thomson AR, Rocca D, Henley JM. The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 2014 Dec 26;289(52):36140-9.
Mouse anti Human protein gene product 9.5 antibody, clone 31A3 (7863-1004) used to identify PGP9.5 expressing canine gonadal cells transplanted into nude mice by immunohistochemistry on formalin fixed, paraffin embedded tissue sections.
Image caption:
Histological and immunohistochemical analyses of 4- and 5-month-old BM testes.
Panels (a,c) represent hematoxylin and eosin-stained testis sections in 4- and 5-month-old BMs. Testis sections of 4- (b) and 5-month-old (d) BMs were stained with anti-PGP9.5 antibody. Black arrowheads indicate PGP9.5-positive germ cells. Scale bars are 20 μm in all panels. GC, germ cell; IC, interstitial cell; SC, Sertoli cell. Magnification is 400× in all panels. Gonadal somatic index (GSI) = [Gonad Weight/Total Tissue Weight] × 100 (e). (*P<0.05).
From: Lee KH, Lee WY, Kim DH, Lee SH, Do JT, Park C, Kim JH, Choi YS, Song H.
Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice.
Sci Rep. 2016 Feb 24;6:21919.
Mouse anti Human protein gene product 9.5 antibody, clone 31A3 (7863-1004) used to identify PGP9.5 expressing canine gonadal cells in nude mice by immunofluorescence on formalin fixed, paraffin embedded tissue sections.
Image caption:
The detection of BM GDC cells after the transplantation of red fluorescent-labelled GDC cells into the seminiferous tubules of germ cell-depleted immunodeficient mice (12 weeks).
Hematoxylin and eosin staining of busulfan-treated immunodeficient mouse testes (a). Red fluorescent-labelled GDC cells from 4-month-old BM testes (b, left testis) and GDC cells with no labelling (b, right testis) were transplanted into the immunodeficient mouse testes. A bright field image (c) and fluorescent image (d) of seminiferous tubules transplanted with red fluorescent-labelled GDC cells are shown. Panel (e) represents the merged image of panels (c,d). Panel (f) represents (a) bright field image, and panel g represents a fluorescent image of seminiferous tubules transplanted with red fluorescent-labelled GDC cells stained with anti-PGP9.5 antibody. Panel (h) is (a) merged image of panels (f,g). Panels (i,j) represent a bright field image (i) and a fluorescent image (j), respectively, of seminiferous tubules stained with anti-PGP9.5 antibody in a control mouse testis. Panels (l,m) show a bright field image (l) and fluorescent image (m), respectively, of seminiferous tubules without anti-PGP9.5 antibody staining in a control mouse testis. Panel (k) shows (a) merged image of panels (i,j) and panel (n) is (a) merged image of panels (l,m). White and yellow arrows indicate red fluorescent-labeled GDC cells and PGP9.5-positive cells, respectively. Scale bars are 50 mm in panel (b). Scale bars are 50 μm and magnification is 100× in panels a and (c–n).
From: Lee KH, Lee WY, Kim DH, Lee SH, Do JT, Park C, Kim JH, Choi YS, Song H.
Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice.
Sci Rep. 2016 Feb 24;6:21919.
Mouse anti Human protein gene product 9.5 antibody, clone 31A3 (7863-1004) used to identify PGP9.5 expressing canine gonadal cells transplanted into nude mice by immunohistochemistry on formalin fixed, paraffin embedded tissue sections.
Image caption:
Neo-synthesis of BM seminiferous tubules following the xenotransplantation of GDC and testicular somatic cells into dorsal skin (20 weeks).
Mouse xenografted with thawed GDC cells and testicular cells was sacrificed after 20 weeks (a). Panel (b) shows harvested tissues from the inner part of dorsal skin; white and green arrowheads indicate the tissues formed after xenotransplantation (GDC cells and testicular cells) and blood vessels, respectively. Scale bars are 50 mm in panels (a,b). Panel (c) shows hematoxylin and eosin staining of a tissue section in GDC cells and testicular cells xenograft at 20 weeks post-grafting, while panel (d) represents (a) magnified image; white and yellow arrows indicate seminiferous tubules and Sertoli cells, respectively. The tissue sections were stained with anti-PGP9.5 antibody (e), and panel (f) represents (a) merged image of panel e image and nuclear staining image. Panel g is a merged image of panel e and bright field image. Green arrowheads indicate PGP9.5-positive cells. Scale bars are 50 μm in all panels.
From: Lee KH, Lee WY, Kim DH, Lee SH, Do JT, Park C, Kim JH, Choi YS, Song H.
Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice.
Sci Rep. 2016 Feb 24;6:21919.
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 recognizes protein gene product 9.5 (PGP9.5), a ubiquitin hydrolase which is widely expressed in neuronal tissues and represents 1-2% of total soluble brain proteins. PGP9.5, also known as ubiquitin C-terminal hydrolase 1 (UCHL-1), is involved in the regulation of the ubuiquitin pathway.
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 stains neuronal cell bodies and axons in the CNS and periphery, small nerve fibres in peripheral tissues, neuroendocrine cells in the pituitary, thyroid, pancreas and tumours of the DNES.
Clones 31A3 and 13C4 each recognise a different epitope towards the N-terminus of the protein. Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 also recognizes PGP9.5 in other species, including rat and rabbit but evidence suggests it does not bind to PGP9.5 in guinea pigs (Wilson et al. 1988). Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 has been used successfully as a capture reagent with clone 13C4 as a detection reagent in a sandwich ELISA to evaluate contamination of processed meat samples with neuronal tissue (Gaunitz et al. 2009).
Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 stains neuronal cell bodies and axons in the CNS and periphery, small nerve fibres in peripheral tissues, neuroendocrine cells in the pituitary, thyroid, pancreas and tumours of the DNES.
Clones 31A3 and 13C4 each recognise a different epitope towards the N-terminus of the protein. Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 also recognizes PGP9.5 in other species, including rat and rabbit but evidence suggests it does not bind to PGP9.5 in guinea pigs (Wilson et al. 1988). Mouse anti Human Protein Gene Product 9.5 antibody, clone 31A3 has been used successfully as a capture reagent with clone 13C4 as a detection reagent in a sandwich ELISA to evaluate contamination of processed meat samples with neuronal tissue (Gaunitz et al. 2009).
Product Details
- Target Species
- Human
- Species Cross-Reactivity
-
Target Species Cross Reactivity Rat Rabbit Guinea Pig - N.B. Antibody reactivity and working conditions may vary between species.
- Product Form
- Purified IgG - liquid
- Preparation
- Purified IgG prepared by affinity chromatography on Protein A
- Buffer Solution
- Phosphate buffered saline
- Preservative Stabilisers
0.09% Sodium Azide (NaN3) - Carrier Free
- Yes
- Immunogen
- Native, from brain
- Approx. Protein Concentrations
- 1mg/ml
Storage Information
- Storage
- Store at +4oC or at -20oC if preferred.
Storage in frost-free freezers is not recommended.
This product should be stored undiluted. Avoid repeated freezing and thawing as this may denature the antibody. Should this product contain a precipitate we recommend microcentrifugation before use. - Shelf Life
- 18 months from date of despatch.
More Information
- UniProt
- P09936 Related reagents
- Entrez Gene
- UCHL1 Related reagents
- GO Terms
- GO:0005886 plasma membrane
- GO:0004197 cysteine-type endopeptidase activity
- GO:0004221 ubiquitin thiolesterase activity
- GO:0005634 nucleus
- GO:0005737 cytoplasm
- GO:0006511 ubiquitin-dependent protein catabolic process
- GO:0008242 omega peptidase activity
- GO:0016579 protein deubiquitination
- GO:0016874 ligase activity
- GO:0031694 alpha-2A adrenergic receptor binding
- GO:0043130 ubiquitin binding
- GO:0043407 negative regulation of MAP kinase activity
- Regulatory
- For research purposes only
Applications of Protein Gene Product 9.5 antibody
| Application Name | Verified | Min Dilution | Max Dilution |
|---|---|---|---|
| ELISA | 1/500 | 1/2000 | |
| Immunohistology - Paraffin 1 | 1/100 | 1/400 | |
| Western Blotting |
- 1 This antibody is suitable for use on paraffin embedded tissue sections. We do however recommend fixation in 95% ethanol/5% acetic acid for 2-3 hours prior to paraffin embedding. Specimens which have not been fixed in acetic acid/alcohol will require pretreatment using the microwave-citrate buffer method.
Where this product has not been tested for use in a particular technique this does not necessarily exclude its use in such procedures. Suggested working dilutions are given as a guide only. It is recommended that the user titrates the product for use in their own system using the appropriate negative/positive controls.
Copyright © 2018 Bio-Rad Antibodies (formerly AbD Serotec)
Secondary Antibodies Available
Product Specific References
Source Reference
-
Day, I.N.M and Thompson, R.J. (1986) Monoclonal antibodies to the neuroendocrine protein PGP 9.5
Biochemical Society Transactions 14: 350–1.
References for Protein Gene Product 9.5 antibody
-
Wilson, P.O. et al. (1988) The immunolocalization of protein gene product 9.5 using rabbit polyclonal and mouse monoclonal antibodies.
Br J Exp Pathol. 69 (1): 91-104. -
Nonclercq, D. et al. (2002) Phenotypic variations and dynamic topography of transformed cells in an experimental model of diethylstilbestrol-induced renal tumour in male Syrian hamster.
Histochem J. 34 (10): 487-97. -
García-Añoveros, J. et al. (2001) Transport and localization of the DEG/ENaC ion channel BNaC1alpha to peripheral mechanosensory terminals of dorsal root ganglia neurons.
J Neurosci. 21 (8): 2678-86. -
Verhein, K.C. et al. (2011) Three days after a single exposure to ozone, the mechanism of airway hyperreactivity is dependent on substance P and nerve growth factor.
Am J Physiol Lung Cell Mol Physiol. 300: L176-84. -
Caballero, O.L. et al. (2002) Interaction and colocalization of PGP9.5 withJAB1 and p27(Kip1).
Oncogene. 21:3003-10. -
Kondo, T. et al. (2013) Prostaglandin E2 mediates acid-induced heartburn in healthy volunteers.
Am J Physiol Gastrointest Liver Physiol. 304(6):G568-73 -
Mihara, H. et al. (2010) Involvement of TRPV2 activation in intestinal movement through nitric oxide
production in mice.
J Neurosci. 30: 16536-44. -
Forsgren, K. et al. (1999) Regeneration of nerve fibres in the maxillary sinus mucosa after experimental surgery. An immunocytochemical double-labelling study in the rabbit.
Acta Otolaryngol. 119: 486-91. -
Yee, C.L. et al. (2001) "Type III" cells of rat taste buds: immunohistochemical and ultrastructural studies of neuron-specific enolase, protein gene product 9.5, and serotonin.
J Comp Neurol. 440: 97-108. -
Pauza, D.H. et al. (2014) A combined acetylcholinesterase and immunohistochemical method for precise anatomical analysis of intrinsic cardiac neural structures.
Ann Anat. pii: S0940-9602(14)00159-9. -
Sugimoto, K. et al. (2011) Olmesartan ameliorates peripheral nerve dysfunction in Zucker diabetic fatty rats.
J Hypertens. 29: 1337-46. -
Bishop, P. et al. (2014) The ubiquitin C-terminal hydrolase L1 (UCH-L1) C terminus plays a key role in protein stability, but its farnesylation is not required for membrane association in primary neurons.
J Biol Chem. 289 (52): 36140-9. -
Moldovan, M. et al. (2013) Peptide mimetic of the S100A4 protein modulates peripheral nerve regeneration and attenuates the progression of neuropathy in myelin protein P0 null mice.
Mol Med. 19: 43-53. -
Kaleczyc, J. et al. (2007) The distribution and chemical coding of intramural neurons supplying the porcine stomach - the study on normal pigs and on animals suffering from swine dysentery.
Anat Histol Embryol. 36 (3): 186-93. -
Silva, I. et al. (2015) Activation of P2Y6 Receptors Facilitates Nonneuronal Adenosine Triphosphate and Acetylcholine Release from Urothelium with the Lamina Propria of Men with Bladder Outlet Obstruction.
J Urol. 194 (4): 1146-54. -
Gaunitz C et al. (2009) Suitability of antigens PGP 9.5 and neurofilament light as marker proteins for detection of neuronal tissue in processed meat products.
J Food Prot. 72 (5): 1070-7. -
Forsgren, K. et al. (1999) Regeneration of nerve fibres in the maxillary sinus mucosa after experimental surgery. An immunocytochemical double-labelling study in the rabbit.
Acta Otolaryngol. 119 (4): 486-91. -
Pauziene, N. et al. (2016) Innervation of the rabbit cardiac ventricles.
J Anat. 228 (1): 26-46. -
Rashwan, A. et al. (2016) Ontogeny and innervation of taste buds in mouse palatal gustatory epithelium.
J Chem Neuroanat. 71: 26-40. -
Inokaitis, H. et al. (2016) Innervation of sinoatrial nodal cells in the rabbit.
Ann Anat. Apr 1. pii: S0940-9602(16)30039-5. [Epub ahead of print] -
Lee, W-Y. et al. (2016) Establishment of a surgically induced cryptorchidism canine recipient model for spermatogonial stem cell transplantation.
Laboratory Animal Research. 32 (4): 257. -
Lee, K.H. et al. (2016) Vitrified canine testicular cells allow the formation of spermatogonial stem cells and seminiferous tubules following their xenotransplantation into nude mice.
Sci Rep. 6: 21919. -
Lakritz, J.R. et al. (2017) An oral form of methylglyoxal-bis-guanylhydrazone reduces monocyte activation and traffic to the dorsal root ganglia in a primate model of HIV-peripheral neuropathy.
J Neurovirol. May 1 [Epub ahead of print] -
Day, I.N. & Thompson, R.J. (2010) UCHL1 (PGP 9.5): neuronal biomarker and ubiquitin system protein.
Prog Neurobiol. 90 (3): 327-62. -
Park, H.J. et al. (2017) Stage-specific expression of Sal-like protein 4 in boar testicular germ cells.
Theriogenology. 101: 44-52. -
Hur, T.Y. et al. (2017) Dose-dependent effects of busulfan on dog testes in preparation for spermatogonial stem cell transplantation.
Lab Anim Res. 33 (3): 264-9. -
Park, H.J. et al. (2018) Species-specific expression of phosphoglycerate kinase 2 (PGK2) in the developing porcine testis.
Theriogenology. 110: 158-167.
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