Revision 8

#88742

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Orders:

877-616-CELL (2355)

[email protected]

Support:

877-678-TECH (8324)

3 Trask Lane | Danvers | Massachusetts | 01923 | USA

For Research Use Only. Not for Use in Diagnostic Procedures.

Applications:
W, 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 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.

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 involves its function as a key mediator of inflammatory responses (4).

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

W: 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

M: Mouse

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.

限制使用

除非 CST 的合法授书代表以书面形式书行明确同意,否书以下条款适用于 CST、其关书方或分书商提供的书品。 任何书充本条款或与本条款不同的客书条款和条件,除非书 CST 的合法授书代表以书面形式书独接受, 否书均被拒书,并且无效。

专品专有“专供研究使用”的专专或专似的专专声明, 且未专得美国食品和专品管理局或其他外国或国内专管机专专专任何用途的批准、准专或专可。客专不得将任何专品用于任何专断或治专目的, 或以任何不符合专专声明的方式使用专品。CST 专售或专可的专品提供专作专最专用专的客专,且专用于研专用途。将专品用于专断、专防或治专目的, 或专专售(专独或作专专成)或其他商专目的而专专专品,均需要 CST 的专独专可。客专:(a) 不得专独或与其他材料专合向任何第三方出售、专可、 出借、捐专或以其他方式专专或提供任何专品,或使用专品制造任何商专专品,(b) 不得复制、修改、逆向工程、反专专、 反专专专品或以其他方式专专专专专品的基专专专或技专,或使用专品开专任何与 CST 的专品或服专专争的专品或服专, (c) 不得更改或专除专品上的任何商专、商品名称、徽专、专利或版专声明或专专,(d) 只能根据 CST 的专品专售条款和任何适用文档使用专品 , (e) 专遵守客专与专品一起使用的任何第三方专品或服专的任何专可、服专条款或专似专专

Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 8

Cell Signaling Technology Logo
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
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.
Western Blotting Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
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.
Immunoprecipitation Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 8

Cell Signaling Technology Logo
Immunohistochemical analysis of paraffin-embedded mouse ovary using Osteopontin/SPP1 (E9Z1D) Rabbit mAb.
Immunohistochemistry Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
Immunohistochemical analysis of paraffin-embedded mouse pancreas using Osteopontin/SPP1 (E9Z1D) Rabbit mAb.
Immunohistochemistry Image 2: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
Immunohistochemical analysis of paraffin-embedded pons region of mouse brain using Osteopontin/SPP1 (E9Z1D) Rabbit mAb.
Immunohistochemistry Image 3: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 8

Cell Signaling Technology Logo
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 4: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
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 5: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
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.
Immunohistochemistry Image 6: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.

Revision 8

Cell Signaling Technology Logo
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 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
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 2: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
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.
Immunofluorescence Image 1: Osteopontin/SPP1 (E9Z1D) Rabbit mAb
Orders: 877-616-CELL (2355) [email protected] Support: 877-678-TECH (8324) [email protected] Web: cellsignal.com
For Research Use Only. Not for Use in Diagnostic Procedures.