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AF5110-1 Prolyl 4-hydroxylase subunit beta antibody

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Mouse anti Rat Prolyl 4-hydroxylase subunit beta 6-9H6

AF5110-1

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Product Description for Prolyl 4-hydroxylase subunit beta

Mouse anti Rat Prolyl 4-hydroxylase subunit beta 6-9H6.
Presentation: Purified
Product is tested for Frozen Sections, Enzyme Immunoassay, Immunocytochemistry/Immunofluorescence, Western blot / Immunoblot, Paraffin Sections.

Properties for Prolyl 4-hydroxylase subunit beta

Product Category Primary Antibodies
Quantity 0.1 mg
Synonyms Fibroblast marker, P4HB, Prolyl 4-hydroxylase beta
Presentation Purified
Reactivity Rt
Applications C, E, ICC/IF, P, WB
Clonality Monoclonal
Clone 6-9H6
Host Mouse
Isotype IgG1
Shipping to Worldwide
PDF datasheet View Datasheet
Manufacturer OriGene Technologies GmbH
Material safety datasheet MSDS for Monoclonal Antibodies (de)

Datasheet Extract

Immunogen
Immunogen:
Prolyl-4-Hydrolase beta.
The epitope is not further characterized.
Isotype control AM03095PU-N, SM20P (for use in rat samples)
Add. information Has been used successfully as a marker antibody for staining Fibroblasts in Immunohistochemistry applications.
Application ELISA.
Western blot: 2-5 µg/ml (See also Bai, Y. et al. 1986).
Immunofluorescence.
Immunohistochemistry on Frozen Sections: 1 µg/ml (1/200) (See Protocols below).
Immunohistochemistry on Paraffin Sections: 10 µg/ml (1/20), Microwave pretreatment for antigen retrieval is recommended (See Protocols below).
Recommended Positive Control: Rat skin.
Background Collagen Prolyl 4-Hydroxylases (P4H) play an essential role in the synthesis of all collagens. Two alpha and two beta subunits assemble into P4H tetramers in which protein disulfide isomerase (PDI) acts as the beta subunit.
P4HB, Prolyl 4 hydroxylase subunit beta, is a multifunctional and highly abundant enzyme that belongs to the protein disulfide isomerase family. When present as a tetramer consisting of two alpha and two beta subunits, this enzyme is involved in hydroxylation of prolyl residues in preprocollagen. This enzyme is also a disulfide isomerase containing two thioredoxin domains that catalyze the formation, breakage and rearrangement of disulfide bonds.
Concentration 0.2 mg/ml (after reconstitution)
Protocols Frozen Sections
Incubations are done at RT. Water is of double distilled or comparable quality.
1. Fix fresh frozen sections in ice-cold acetone for 10 min 
2. Block endogenous peroxidase with 100ml 0.15M Sodium Azide / 0.15% H2O2 in PBS 
3. Wash in PBS 
4. Block with 10% Normal Goat Serum (Jackson #005-000-121) for 30 min in a humid chamber
5. Incubate with primary antibody Cat.-No AF5110-1 at 1 µg/ml for 1 hour in a humid chamber
6. Wash in PBS 
7. Incubate with secondary antibody (peroxidase-conjugated Goat Anti Mouse IgG (H+L), f.e. Cat.-No R1253HRP) at a 1/200 dilution for 1 hour in a humid chamber
8. Wash in PBS 
9. Incubate with AEC substrate (3-Amino-9-Ethylcarbazol) for 12 min 
10. Wash in PBS 
11. Counterstain with Mayer's hemalum. 

Paraffin Sections
Incubations are done at RT. Water is of double distilled or comparable quality.
1. Rehydrate paraffin sections
2. Put the slides in a cuvette with 250 ml 0.01 M citrate buffer pH 6.0
3. Heat the slides in a microwave oven for 2 x 7 min and 700Watt
4. Leave the slides in the buffer for 20 min
5. Block endogenous peroxidase with 1%H2O2 in water
6. Wash in PBS
7. Block with 10% Normal Goat Serum (Jackson #005-000-121) for 30 min in a humid chamber
8. Incubate with primary antibody Cat.-No AF5110-1 at 10 µg/ml for 1 hour in a humid chamber
9. Wash in PBS
10. Incubate with secondary antibody (peroxidase-conjugated Goat Anti Mouse IgG (H+L), f.e Cat.-No R1253HRP) at a 1/200 dilution for 1 hour in a humid chamber
11. Wash in PBS
12. Incubate with AEC substrate (3-Amino-9-Ethylcarbazol) for 12 min 
13. Wash in PBS
14. Counterstain

Immunofluorescence
1. Wash with PBS
2. Fixation using 3.7% Formaldehyde for 10 min
3. Blocking using PBS containing 0.2% Triton X-100 and 1% BSA for 10 min
4. Incubation with 1. Ab (Mouse anti-Rat anti Prolyl-4-Hydroxylase-beta Cat.-No AF5110-1) for 1 hour
5. 3x wash for 5 min using PBS containing 0.2% Triton X-100
6. Inkubation wth 2. Ab (FITC-labeled Goat anti-Mouse, Cat.-No R1253F) for 1 hour
7. 3x wash for 5 min using PBS containing 0.2% Triton X-100.
Product Citations
Purchased from Acris:
1. Zheng W, Christensen LP, Tomanek RJ. Differential effects of cyclic and static stretch on coronary microvascular endothelial cell receptors and vasculogenic/angiogenic responses. Am J Physiol Heart Circ Physiol. 2008 Aug;295(2):H794-800. doi: 10.1152/ajpheart.00343.2008. Epub 2008 Jun 27. PubMed PMID: 18586890.
2. Gruh I, Beilner J, Blomer U, Schmiedl A, Schmidt-Richter I, Kruse ML, et al. No evidence of transdifferentiation of human endothelial progenitor cells into cardiomyocytes after coculture with neonatal rat cardiomyocytes. Circulation. 2006 Mar 14;113(10):1326-34. Epub 2006 Mar 6. PubMed PMID: 16520414.
3. Hahn JY, Cho HJ, Bae JW, Yuk HS, Kim KI, Park KW, et al. Beta-catenin overexpression reduces myocardial infarct size through differential effects on cardiomyocytes and cardiac fibroblasts. J Biol Chem. 2006 Oct 13;281(41):30979-89. Epub 2006 Aug 18. PubMed PMID: 16920707.
4. Koleganova N, Piecha G, Ritz E, Gross ML. Calcitriol ameliorates capillary deficit and fibrosis of the heart in subtotally nephrectomized rats. Nephrol Dial Transplant. 2009 Mar;24(3):778-87. doi: 10.1093/ndt/gfn549. Epub 2008 Oct 1. PubMed PMID: 18829613.
5. Kapur NK, Deming CB, Kapur S, Bian C, Champion HC, Donahue JK, et al. Hemodynamic modulation of endocardial thromboresistance. Circulation. 2007 Jan 2;115(1):67-75. Epub 2006 Dec 26. PubMed PMID: 17190863.
6. Haylor J, Schroeder J, Wagner B, Nutter F, Jestin G, Idée JM, et al. Skin gadolinium following use of MR contrast agents in a rat model of nephrogenic systemic fibrosis. Radiology. 2012 Apr;263(1):107-16. doi: 10.1148/radiol.12110881. Epub 2012 Feb 17. PubMed PMID: 22344402. 
7. Nishida, E;Miyaji, H;Kato, A;Takita, H;Iwanaga, T;Momose, T;Ogawa, K;Murakami, S;Sugaya, T;Kawanami, M;2016Graphene oxide scaffold accelerates cellular proliferative response and alveolar bone healing of tooth extraction socket. Int J Nanomedicine 2016. 2265-77, 11. PubMed PMID: 27307729. 
8. Edanami, N;Yoshiba, N;Ohkura, N;Takeuchi, R;Tohma, A;Noiri, Y;Yoshiba, K;2017Characterization of Dental Pulp Myofibroblasts in Rat Molars after PulpotomyJ Endod 2017. PubMed PMID: 28527846. 
9. Murakami, S;Miyaji, H;Nishida, E;Kawamoto, K;Miyata, S;Takita, H;Akasaka, T;Fugetsu, B;Iwanaga, T;Hongo, H;Amizuka, N;Sugaya, T;Kawanami, M. Dose effects of beta-tricalcium phosphate nanoparticles on biocompatibility and bone conductive ability of three-dimensional collagen scaffolds. Dent Mater J 2017. PubMed PMID: 28450672.
General Readings
  1. Bai Y, Muragaki Y, Obata K, Iwata K, Ooshima A. Immunological properties of monoclonal antibodies to human and rat prolyl 4-hydroxylase. J Biochem. 1986 Jun;99(6):1563-70. PubMed PMID: 3017922.
  2. Okuno M, Muto Y, Kato M, Moriwaki H, Noma A, Tagaya O, et al. Changes in serum and hepatic levels of immunoreactive prolyl hydroxylase in two models of hepatic fibrosis in rats. J Gastroenterol Hepatol. 1991 May-Jun;6(3):271-7. PubMed PMID: 1655096.
Storage Prior to reconstitution store at 2-8°C.
Following reconstitution store undiluted at 2-8°C for one month
or (in aliquots) at -20°C for longer.
Avoid repeated freezing and thawing.
Shelf life: one year from despatch.
Format
Purification:
Affinity Chromatography on Protein A
Buffer System:
PBS, pH 7.2
Preservatives:
0.09% Sodium Azide
Stabilizers:
0.5% BSA
State:
Lyophilized purified IgG fraction
Purified
Reconstitution:
Restore in 0.5 ml double distilled water
Species Reactivity
Species reactivity (tested):
Rat.
Specificity
Specificity:
This antibody specifically reacts with the beta-subunit of Rat P4H (Fibroblast Marker) from the skin of newborn Rat.
Does not react with Mouse according to customer information.

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