Osteopontin Antikörper (Secreted phosphoprotein 1)

Details for Product anti-SPP1 Antibody No. ABIN105163
Antigen
  • BNSP
  • BSPI
  • ETA-1
  • OPN
  • 2AR
  • Apl-1
  • Bsp
  • Eta
  • OP
  • Opn
  • Opnl
  • Ric
  • Spp-1
  • OSP
  • zgc:111821
  • secreted phosphoprotein 1
  • SPP1
  • Spp1
  • spp1
Reaktivität
Human
373
111
74
23
9
5
4
4
4
2
1
1
1
Wirt
Kaninchen
253
153
24
8
1
Klonalität
Polyklonal
Konjugat
Dieser Osteopontin Antikörper ist unkonjugiert
30
21
19
12
11
7
6
6
6
6
6
6
2
2
2
2
2
1
1
1
Applikation
Immunohistochemistry (Formalin-fixed Paraffin-embedded Sections) (IHC (fp)), Immunohistochemistry (IHC), ELISA, Western Blotting (WB)
344
232
128
58
26
24
22
20
19
18
17
13
3
2
2
2
1
Optionen
'Independent Validation' Siegel
Antigen SPP1
Chargennummer 27944
Validierte Anwendung Western Blotting
Positivkontrolle

human pluripotent stem cells (day 0)

pancreatic endoderm cells (day 10)

pancreatic progenitor cells (day 14)

Negativkontrolle

definite endoderm cells (day 4)

Bewertung

ABIN1043686 reveals two protein bands of the expected size and some weaker extraneous bands in cell lysates of human pluripotent stem cells, definite endoderm cells, pancreatic endoderm cells, and pancreatic progenitor cells.

Primärantikörper ABIN1043686
Sekundärantikörper donkey anti-rabbit HRP-conjugated antibody (GE Healthcare, NA9310V)
Protokoll
  • Grow HUES8 in mTeSR1 (STEMCELL Technologies, 85850) at 37°C and 5% O2, 5% CO2 in 2ml in a 6-well plate to 90% confluency.
  • Harvest cells using TrypLE (ThermoFisher Scientific, 12604013) following the manufacturer´s instructions.
  • Lyse 2x106 cells in 30µl per well cold RIPA buffer (50mM Tris (pH 8.0), 150mM NaCl, 0.1% SDS, 0.5% deoxycholate, 1% Triton X-100 in ddH2O) supplemented with 1mM PMSF and 1x protease inhibitor (Roche, 11836170001) for 30min on ice with 3x vortexing in 1.5ml microcentrifuge tubes.
  • Centrifuge tubes at 10000xg for 8min at 4°C.
  • Transfer supernatant to a new 1.5ml microcentrifuge tube and store at -80°C.
  • Determine total protein content of the lysates using a Bradford assay (Bio-Rad, 500-0006).
  • Denature 50µg of total protein for 5min at 95°C in 30µl 1x Laemmli SDS sample buffer and subsequently separate them on a denaturing 7.5% polyacrylamide gel (7.5% Acrylamide, 0.375M Tris-HCl pH8.8, 0.1% SDS, 0.1% APS, 0.1% TEMED) in an electrophoresis chamber for approximately 90min at 120V.
  • Transfer proteins onto a PVDF membrane (Sigma Aldrich, IPVH00010) with transfer buffer (5.27g Tris, 2.93 g glycerine, 200 ml methanol, fill to 1l ddH2O) in a semidry western blotting system for 75min at 80mA/gel.
  • Check transfer of the separated proteins by Ponceau S staining.
  • Rinse membrane with water.
  • Wash membrane for 5min with TBST (TBS, 0.02% Tween20).
  • Block the membrane with 20ml blocking buffer (TBST, 5% milk) on a shaker for 1h at RT.
  • Rinse membrane 3x with TBST.
  • Wash membrane on a shaker 3x for 5min with TBST.
  • Shrink-wrap and incubate membrane with primary
    • rabbit anti-osteopontin antibody (antibodies-online, ABIN1043686, lot 27944) diluted 1:1000 and 1:10000 respectively in blocking buffer ON at 4°C or
    • mouse anti-beta actin antibody (Sigma Aldrich, A1978) diluted 1:2000 in blocking buffer at RT for 1h.
  • Wash membrane 3x for 5min with TBST.
  • Incubate membrane with secondary
    • donkey anti-rabbit HRP-conjugated antibody (GE Healthcare, NA9310V) diluted 1:5000 in TBST containing 1% milk for 1h at RT or
    • donkey anti-mouse HRP-conjugated antibody (GE Healthcare, NA9340V) diluted 1:5000 in TBST containing 1% milk for 1h at RT.
  • Wash membrane 3x for 5min with TBST.
  • Reveal protein bands using ECL solution (ThermoScientific, 34076) on a Fusion SL (Vilber) chemiluminescence system. Exposure times for the 1:1000 and 1:10000 dilutions of ABIN1043686 were 30sec and 5min respectively.
Anmerkungen
  • ABIN1043686 revealed some unspecific bands. Specificity of the two strongest bands at 66kDa and 32kDa is likely, although it has to be validated in knockout or knockdown model to be certain. The cleaved band at 32kDa might decrease during differentiation in line with transcriptomic data, the full-length protein at 66kDa might be also strongest expressed in pluripotent stem cells (lanes 1 and 2). However, signal intensity in lysates from definite endoderm cells (lanes 3 and 4), pancreatic endoderm cells (lanes 5 and 6), and pancreatic progenitors (lanes 7 and 8) was higher than expected. The signal appears to be the strongest for pancreatic progenitor samples (land 8). This is also true for the corresponding loading control, suggesting that more total protein was loaded.

  • The number of the extraneous protein bands is reduced when using ABIN1043686 at a higher dilution of 1:10000 for the western blot.

  • ABIN1043686 was also tested in IHC on 4-5µm FFPE sections of primary human kidney cancer tissue. Epitope retrieval was carried out using Tris-EDTA buffer at pH9.0 (Zytomed, ZUC029-500), EDTA at pH8.0 (Leica, RE7116), or citrate buffer at pH6.1 (Agilent, S169984-2) for 20min in a decloaking chamber. Recommendation for antigen retrieval no pre-treatment, which did not give any bad signal. Comparing SPP1 the observed staining pattern with Human Protein Atlas expression data indicates that the staining pattern using the citrate buffer at pH6.0 looks most convincing. Validation in knockout models is necessary to clarify without doubts.

Validierungsbilder
Western Blotting validation image for anti-Secreted phosphoprotein 1 (SPP1) antibody (ABIN1043686) Western blot with ABIN1043686 on human pluripotent stem cells (1 and 2), definite end...
Immunogen This whole rabbit serum was prepared by repeated immunizations with a synthetic peptide, from the human osteopontin protein, conjugated to KLH using maleimide.
Produktmerkmale Concentration Definition: by Refractometry
Reinigung Antiserum
Plasmids, Primers & others Plasmide, Primers & weitere Osteopontin products on genomics-online (e.g. as negative or positive controls)
Antigen
Andere Bezeichnung Osteopontin (SPP1 Antibody Abstract)
Hintergrund Anti Osteopontin Antibody recognizes Osteopontin (OPN) that is an arginine-glycine-aspartic acid (RGD)-containing glycoprotein which interacts with integrins and CD44 as major receptors. OPN is multifunctional, with activities in cell migration, cell survival, inhibition of calcification, regulation of immune cell function, and control of tumor cell phenotype. The gene encoding OPN is called spp1.  Targeting this gene has revealed that while OPN is not necessary for normal embryonic development, fertility and health under pathogen-free conditions, loss of the protein has significant consequences in several models of injury/disease as diverse as renal injury, viral and bacterial infection, bone remodeling, and tumor growth. The fact that no other proteins seem to share a redundant activity with OPN under these conditions suggests that OPN has a unique functional role during tissue injury and stress. Interestingly, several members of the matrix metalloproteinase (MMP) family are also induced during injury/disease processes in patterns overlapping that of OPN.  OPN has recently been shown to be a novel substrate for two MMPs, MMP-3 (stromelysin-1) and MMP-7 (matrilysin). There are three cleavage sites for MMP-3 in human OPN, two of which are also cleaved by MMP-7 (see cleavage diagram). Biological assays demonstrate that the MMP-cleaved OPN has increased activity in promoting both cell adhesion and migration compared with full-length OPN. In addition, inhibitory reagents were used to show that the same receptors that interact with OPN also mediate interaction of MMP-cleaved OPN with tumor cells.  It is suggested that active forms of OPN at sites of tissue injury may be regulated by the activity of proteases including MMPs and that the differences in activity of modified OPN may be explained by differences in binding affinity of integrins or distinct downstream signaling events.
Synonyms: Osteopontin Bone sialoprotein 1 Secreted phosphoprotein 1 SPP-1 Urinary stone protein Nephropontin Uropontin SPP1 BNSP, OPN ORF Name: PSEC0156
Gen-ID 6696
UniProt P10451
Pathways Regulation of Cell Size
Applikations-hinweise This antibody is suitable for western blotting, immunohistochemistry (formalin-fixed paraffin-embedded sections) and ELISA. The antibody exclusively recognizes C-terminal fragments of both thrombin and MMP-cleaved OPN.  A 1:1000 dilution will detect strongly approximately 250 ng of OPN protein on a blot. No pretreatment is required for IHC when formalin-fixed paraffin-embedded tissue is stained.
Beschränkungen Nur für Forschungszwecke einsetzbar
Format Liquid
Konzentration 75 mg/mL
Buffer 0.02 M Potassium Phosphate, 0.15 M Sodium Chloride, pH 7.2
Konservierungs-mittel Sodium azide
Vorsichtsmaßnahmen This product contains sodium azide: a POISONOUS AND HAZARDOUS SUBSTANCE which should be handled by trained staff only.
Lagerung -20 °C
Bilder des Herstellers
Immunohistochemistry (IHC) image for anti-Secreted phosphoprotein 1 (SPP1) antibody (ABIN105163) Rabbit anti-osteopontin was used at a 1:100-1:300 dilution to detect osteopontin by i...
Western Blotting (WB) image for anti-Secreted phosphoprotein 1 (SPP1) antibody (ABIN105163) Rabbit anti-osteopontin was used at a 1:1,000 dilution to detect Osteopontin by Weste...
 image for anti-Secreted phosphoprotein 1 (SPP1) antibody (ABIN105163) anti-Secreted phosphoprotein 1 (SPP1) antibody (Image 3)
Produkt verwendet in: Cherepanova, Gomez, Shankman, Swiatlowska, Williams, Sarmento, Alencar, Hess, Bevard, Greene, Murgai, Turner, Geng, Bekiranov, Connelly, Tomilin, Owens: "Activation of the pluripotency factor OCT4 in smooth muscle cells is atheroprotective." in: Nature medicine, Vol. 22, Issue 6, pp. 657-65, 2017 (PubMed).

Sasaki, Matsumoto, Egusa, Matsusaki, Nishiguchi, Nakano, Akashi, Imazato, Yatani: "In vitro reproduction of endochondral ossification using a 3D mesenchymal stem cell construct." in: Integrative biology : quantitative biosciences from nano to macro, 2012 (PubMed).

Robertson, Bonsal, Chellaiah: "Regulation of Erk1/2 activation by osteopontin in PC3 human prostate cancer cells." in: Molecular cancer, Vol. 9, pp. 260, 2010 (PubMed).

Robertson, Chellaiah: "Osteopontin induces beta-catenin signaling through activation of Akt in prostate cancer cells." in: Experimental cell research, Vol. 316, Issue 1, pp. 1-11, 2009 (PubMed).

Iwanaga, Ueno, Ueki, Huang, Tomita, Okamoto, Ogawa, Ueda, Maekawa, Sakamoto: "The expression of osteopontin is increased in vessels with blood-brain barrier impairment." in: Neuropathology and applied neurobiology, Vol. 34, Issue 2, pp. 145-54, 2008 (PubMed).

Kim, Shin: "Immunohistochemical study of osteopontin in boar testis." in: Journal of veterinary science, Vol. 8, Issue 2, pp. 107-10, 2007 (PubMed).

Shin, Ahn, Kim, Moon, Kang, Lee, Sim, Hyun: "Temporal expression of osteopontin and CD44 in rat brains with experimental cryolesions." in: Brain research, Vol. 1041, Issue 1, pp. 95-101, 2005 (PubMed).

Allgemeine Veröffentlichungen Agnihotri, Crawford, Haro, Matrisian, Havrda, Liaw: "Osteopontin, a novel substrate for matrix metalloproteinase-3 (stromelysin-1) and matrix metalloproteinase-7 (matrilysin)." in: The Journal of biological chemistry, Vol. 276, Issue 30, pp. 28261-7, 2001 (PubMed).

Ashkar, Weber, Panoutsakopoulou, Sanchirico, Jansson, Zawaideh, Rittling, Denhardt, Glimcher, Cantor: "Eta-1 (osteopontin): an early component of type-1 (cell-mediated) immunity." in: Science (New York, N.Y.), Vol. 287, Issue 5454, pp. 860-4, 2000 (PubMed).

McCawley, Matrisian: "Matrix metalloproteinases: multifunctional contributors to tumor progression." in: Molecular medicine today, Vol. 6, Issue 4, pp. 149-56, 2000 (PubMed).

Ophascharoensuk, Giachelli, Gordon, Hughes, Pichler, Brown, Liaw, Schmidt, Shankland, Alpers, Couser, Johnson: "Obstructive uropathy in the mouse: role of osteopontin in interstitial fibrosis and apoptosis." in: Kidney international, Vol. 56, Issue 2, pp. 571-80, 1999 (PubMed).

Noiri, Dickman, Miller, Romanov, Romanov, Shaw, Chambers, Rittling, Denhardt, Goligorsky: "Reduced tolerance to acute renal ischemia in mice with a targeted disruption of the osteopontin gene." in: Kidney international, Vol. 56, Issue 1, pp. 74-82, 1999 (PubMed).

Nau, Liaw, Chupp, Berman, Hogan, Young: "Attenuated host resistance against Mycobacterium bovis BCG infection in mice lacking osteopontin." in: Infection and immunity, Vol. 67, Issue 8, pp. 4223-30, 1999 (PubMed).

Yoshitake, Rittling, Denhardt, Noda: "Osteopontin-deficient mice are resistant to ovariectomy-induced bone resorption." in: Proceedings of the National Academy of Sciences of the United States of America, Vol. 96, Issue 14, pp. 8156-60, 1999 (PubMed).

Liaw, Birk, Ballas, Whitsitt, Davidson, Hogan: "Altered wound healing in mice lacking a functional osteopontin gene (spp1)." in: The Journal of clinical investigation, Vol. 101, Issue 7, pp. 1468-78, 1998 (PubMed).

Rittling, Matsumoto, McKee, Nanci, An, Novick, Kowalski, Noda, Denhardt: "Mice lacking osteopontin show normal development and bone structure but display altered osteoclast formation in vitro." in: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 13, Issue 7, pp. 1101-11, 1998 (PubMed).

Crawford, Matrisian, Liaw: "Distinct roles of osteopontin in host defense activity and tumor survival during squamous cell carcinoma progression in vivo." in: Cancer research, Vol. 58, Issue 22, pp. 5206-15, 1998 (PubMed).

Weber, Cantor: "The immunology of Eta-1/osteopontin." in: Cytokine & growth factor reviews, Vol. 7, Issue 3, pp. 241-8, 1997 (PubMed).

Uede, Katagiri, Iizuka, Murakami: "Osteopontin, a coordinator of host defense system: a cytokine or an extracellular adhesive protein?" in: Microbiology and immunology, Vol. 41, Issue 9, pp. 641-8, 1997 (PubMed).

Denhardt, Lopez, Rollo, Hwang, An, Walther: "Osteopontin-induced modifications of cellular functions." in: Annals of the New York Academy of Sciences, Vol. 760, pp. 127-42, 1995 (PubMed).

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