Revision 7
Cell Signaling Technology

Orders: 877-616-CELL (2355) [email protected]

Support: 877-678-TECH (8324)

Web: [email protected] cellsignal.com

3 Trask LaneDanversMassachusetts01923USA
For Research Use Only. Not for Use in Diagnostic Procedures.
Applications:

WB, W-S, IP, IHC-P, IF-F, IF-IC, FC-FP

REACTIVITY:

M

SENSITIVITY:

Endogenous

MW (kDa):

58-75

Source/Isotype:

Rabbit IgG

UniProt ID:

#P10923

Entrez-Gene Id:

20750

Product Information

Product Usage Information

Application Dilution
Western Blotting 1:1000
Simple Western™ 1:10 - 1:50
Immunoprecipitation 1:200
Immunohistochemistry (Paraffin) 1:50 - 1:200
Immunofluorescence (Frozen) 1:200 - 1:800
Immunofluorescence (Immunocytochemistry) 1:200 - 1:800
Flow Cytometry (Fixed/Permeabilized) 1:200 - 1:800

Storage

Supplied in 10 mM sodium HEPES (pH 7.5), 150 mM NaCl, 100 µg/mL BSA, 50% glycerol, and less than 0.02% sodium azide. Store at –20°C. Do not aliquot the antibody.

For a carrier free (BSA and azide free) version of this product see product #17090.

Specificity / Sensitivity

Osteopontin/SPP1 (E9Z1D) Rabbit mAb recognizes endogenous levels of total osteopontin/SPP1 protein.

Species Reactivity:

Mouse

Source / Purification

Monoclonal antibody is produced by immunizing animals with a synthetic peptide corresponding to residues surrounding Ala149 of mouse osteopontin/SPP1 protein.

Background

Osteopontin is a highly phosphorylated secreted glycoprotein expressed in a variety of cell types. Osteopontin, encoded by the gene secreted phosphoprotein 1 (SPP1), was originally isolated and cloned from a rat osteosarcoma cell line (1). Osteopontin is a highly negatively charged protein that likely regulates extracellular matrix turnover and remodeling, both in bone and other tissue (2). Osteopontin also has three highly conserved receptor-binding domains, otherwise known as arginine-glycine-aspartic acid (RGD) domains. Binding of osteopontin to cell surface integrins likely activates cell signaling pathways that regulate cell proliferation, adhesion, invasion, migration, and fibrosis (3,4). Osteopontin may be regulated by posttranslational regulation, including phosphorylation, glycosylation, and proteolytic cleavage (5). Osteopontin is also linked to several human diseases. For example, cleaved osteopontin is higher in patients suffering with cirrhosis, a disease involving fibrosis of the liver (5). Its role in the brain is unclear, but SPP1 expression in microglia, the resident macrophages of the brain, is upregulated in mouse neurodegenerative disease models, including Alzheimer’s disease (6). Osteopontin’s role in disease likely involve its function as a key mediator of inflammatory responses (4).

  1. Oldberg, A. et al. (1986) Proc Natl Acad Sci U S A 83, 8819-23.
  2. Kazanecki, C.C. et al. (2007) J Cell Biochem 102, 912-24.
  3. Standal, T. et al. (2004) Exp Oncol 26, 179-84.
  4. Lamort, A.S. et al. (2019) Cells 8, 815. doi: 10.3390/cells8080815.
  5. Song, Z. et al. (2021) Hepatology 73, 1594-1608.
  6. Sala Frigerio, C. et al. (2019) Cell Rep 27, 1293-1306.e6.

Species Reactivity

Species reactivity is determined by testing in at least one approved application (e.g., western blot).

Western Blot Buffer

IMPORTANT: For western blots, incubate membrane with diluted primary antibody in 5% w/v BSA, 1X TBS, 0.1% Tween® 20 at 4°C with gentle shaking, overnight.

Applications Key

WB: Western Blotting W-S: Simple Western™ IP: Immunoprecipitation IHC-P: Immunohistochemistry (Paraffin) IF-F: Immunofluorescence (Frozen) IF-IC: Immunofluorescence (Immunocytochemistry) FC-FP: Flow Cytometry (Fixed/Permeabilized)

Cross-Reactivity Key

H: human M: mouse R: rat Hm: hamster Mk: monkey Vir: virus Mi: mink C: chicken Dm: D. melanogaster X: Xenopus Z: zebrafish B: bovine Dg: dog Pg: pig Sc: S. cerevisiae Ce: C. elegans Hr: horse GP: Guinea Pig Rab: rabbit All: all species expected

Trademarks and Patents

Cell Signaling Technology is a trademark of Cell Signaling Technology, Inc.
Alexa Fluor is a registered trademark of Life Technologies Corporation.
All other trademarks are the property of their respective owners. Visit cellsignal.com/trademarks for more information.

使用に関する制限

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Revision 7
#88742

Osteopontin/SPP1 (E9Z1D) Rabbit mAb

Western Blotting Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Western blot analysis of extracts from RAW 264.7 and Neuro-2a cells, untreated (-) or treated with λ phosphatase (+), using Osteopontin/SPP1 (E9Z1D) Rabbit mAb (upper) or β-Actin (D6A8) Rabbit mAb #8457 (lower).
Western Blotting Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Simple Western™ analysis of lysates (0.1 mg/mL) from Raw 264.7 cells treated with Lambda using Osteopontin/SPP1 (E9Z1D) Rabbit mAb #88742. The virtual lane view (left) shows the target band (as indicated) at 1:10 and 1:50 dilutions of primary antibody. The corresponding electropherogram view (right) plots chemiluminescence by molecular weight along the capillary at 1:10 (blue line) and 1:50 (green line) dilutions of primary antibody. This experiment was performed under reducing conditions on the Jess™ Simple Western instrument from ProteinSimple, a BioTechne brand, using the 12-230 kDa separation module.
Immunoprecipitation Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Immunoprecipitation of osteopontin/SPP1 protein from RAW 264.7 cell extracts. Lane 1 is 10% input, lane 2 is Rabbit (DA1E) mAb IgG XP® Isotype Control #3900, and lane 3 is Osteopontin/SPP1 (E9Z1D) Rabbit mAb. Western blot analysis was performed using Osteopontin/SPP1 (E4O2F) Rabbit mAb #27927. Mouse Anti-Rabbit IgG (Light-Chain Specific) (D4W3E) mAb (HRP Conjugate) #93702 was used as a secondary antibody.
Immunohistochemistry Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse ovary using Osteopontin/SPP1 (E9Z1D) Rabbit mAb.
Immunohistochemistry Image 2: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse pancreas using Osteopontin/SPP1 (E9Z1D) Rabbit mAb.
Immunohistochemistry Image 3: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded pons region of mouse brain using Osteopontin/SPP1 (E9Z1D) Rabbit mAb.
Immunohistochemistry Image 4: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded renca syngeneic tumor (left), 4T1 syngeneic tumor (middle), or mouse liver (right) using Osteopontin/SPP1 (E9Z1D) Rabbit mAb (top) or an Osteopontin/SPP1 Rabbit mAb (bottom). These two antibodies detect unique, non-overlapping epitopes on mouse Osteopontin/SPP1. The similar staining patterns obtained with both antibodies help to confirm the specificity of the staining.
Immunohistochemistry Image 5: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded mouse kidney using Osteopontin/SPP1 (E9Z1D) Rabbit mAb (left) compared to concentration-matched Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 (right). 
Immunohistochemistry Image 6: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Immunohistochemical analysis of paraffin-embedded RAW 264.7 cell pellet (left, positive) or Neuro-2a cell pellet (right, negative) using Osteopontin/SPP1 (E9Z1D) Rabbit mAb.
Immunofluorescence Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of fixed frozen mouse kidney labeled with Osteopontin/SPP1 (E9Z1D) Rabbit mAb (left, green). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with Ras (E4K9L) Rabbit mAb (Alexa Fluor® 488 Conjugate) #51813 (right, red pseudocolor) and DAPI #4083 (right, blue).
Immunofluorescence Image 2: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of fixed frozen mouse thalamus labeled with Osteopontin/SPP1 (E9Z1D) Rabbit mAb (left, green). Free secondary binding sites were then blocked with Rabbit (DA1E) mAb IgG XP® Isotype Control #3900 prior to co-labeling with Iba1/AIF-1 (E4O4W) XP® Rabbit mAb (Alexa Fluor® 647 Conjugate) #78060 (right, red) and DAPI #4083 (right, blue).
Immunofluorescence Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb Expand Image
Confocal immunofluorescent analysis of RAW 264.7 cells (left, positive) and Neuro-2a cells (right, negative) using Osteopontin/SPP1 (E9Z1D) Rabbit mAb (green) and DAPI #4083 (blue). Cells were treated with Brefeldin A #9972 (100 ng/mL, 90 min) to inhibit protein secretion.
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