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Osteopontin/SPP1 Recombinant Rabbit Monoclonal Antibody [PSH14-11] - BSA and Azide free

Usd: 649

Specification

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Catalog# HA751506

Osteopontin/SPP1 Recombinant Rabbit Monoclonal Antibody [PSH14-11] - BSA and Azide free

Application

  • IHC-P

  • IHC-Fr

  • WB

  • IF-Cell

Reactivity

  • Mouse

  • Rat

With BSA and Azide
Conjugation

  • unconjugated

Overview

Product Name

Osteopontin/SPP1 Recombinant Rabbit Monoclonal Antibody [PSH14-11] - BSA and Azide free

Antibody Type

Recombinant Rabbit monoclonal Antibody

Immunogen

Recombinant protein within mouse Osteopontin aa 1-294.

Species Reactivity

Mouse, Rat

Validated Applications

IHC-P, IHC-Fr, WB, IF-Cell

Target Molecular Weight

Predicted band size: 32 kDa

Positive Control

Mouse knee joint tissue, RAW264.7 treated with 100ng/mL LPS for 4 hours add 1μg/mL BFA for last 3 hours cell lysate, RAW264.7 add 100nM TPA overnight then treated with 100ng/mL LPS for 4 hours add 1μg/mL BFA for last 3 hours cell lysate, RAW264.7 cells treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours.

Conjugation

unconjugated

Clone Number

PSH14-11

Product Features

Form

Liquid

Concentration

1mg/ml

Storage Instructions

Store at +4℃ after thawing. Aliquot store at -20℃. Avoid repeated freeze / thaw cycles.

Storage Buffer

1*PBS (pH7.4).

Isotype

IgG

Purification Method

Protein A affinity purified.

Application Dilution

  • IHC-P

  • 1:1,000-1:9,000

  • IHC-Fr

  • 1:500

  • WB

  • 1:5,000

  • IF-Cell

  • 1:2,000

Target

Function

Osteopontin (OPN), also known as bone /sialoprotein I (BSP-1 or BNSP), early T-lymphocyte activation (ETA-1), secreted phosphoprotein 1 (SPP1), 2ar and Rickettsia resistance (Ric), is a protein that in humans is encoded by the SPP1 gene (secreted phosphoprotein 1). The murine ortholog is Spp1. Osteopontin is a SIBLING (glycoprotein) that was first identified in 1986 in osteoblasts. The prefix osteo- indicates that the protein is expressed in bone, although it is also expressed in other tissues. The suffix -pontin is derived from “pons,” the Latin word for bridge, and signifies osteopontin's role as a linking protein. Osteopontin is an extracellular structural protein and therefore an organic component of bone. The gene has 7 exons, spans 5 kilobases in length and in humans it is located on the long arm of chromosome 4 region 22 (4q1322.1). The protein is composed of ~300 amino acids residues and has ~30 carbohydrate residues attached, including 10 sialic acid residues, which are attached to the protein during post-translational modification in the Golgi apparatus. The protein is rich in acidic residues: 30-36% are either aspartic or glutamic acid.

Background References

1. Han H et al. Macrophage-derived Osteopontin (SPP1) Protects From Nonalcoholic Steatohepatitis. Gastroenterology. 2023 Jul

2. Shirakawa K et al. Osteopontin in Cardiovascular Diseases. Biomolecules. 2021 Jul

Subcellular Location

Secreted.

UNIPROT

SwissProt: P10923 Mouse

SwissProt: P08721 Rat

Synonyms

BNSP antibody

Bone sialoprotein 1 antibody

Bone sialoprotein I antibody

BSP I antibody

BSPI antibody

Early T lymphocyte activation 1 antibody

ETA 1 antibody

ETA1 antibody

MGC110940 antibody

Nephropontin antibody

Expand

Images

  • Immunohistochemical analysis of paraffin-embedded mouse knee joint tissue with Rabbit anti-Osteopontin/SPP1 antibody (<a href="/products/HA751506" style="font-weight: bold;text-decoration: underline;">HA751506</a>) at 1/9,000 dilution.<br /><br />The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH<sub>2</sub>O and PBS, and then probed with the primary antibody (<a href="/products/HA751506" style="font-weight: bold;text-decoration: underline;">HA751506</a>) at 1/9,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

    Immunohistochemical analysis of paraffin-embedded mouse knee joint tissue with Rabbit anti-Osteopontin/SPP1 antibody (HA751506) at 1/9,000 dilution.

    The section was pre-treated using heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0) for 20 minutes. The tissues were blocked in 1% BSA for 20 minutes at room temperature, washed with ddH2O and PBS, and then probed with the primary antibody (HA751506) at 1/9,000 dilution for 1 hour at room temperature. The detection was performed using an HRP conjugated compact polymer system. DAB was used as the chromogen. Tissues were counterstained with hematoxylin and mounted with DPX.

  • Application: Immunohistochemistry (IHC-P)<br /><br />Species: Mouse<br />Tissue: Alveolar bone<br />Sample: Paraffin-embedded section<br /><br />Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.<br /><br />Wash buffer: 1× TBST<br />Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.<br />Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.<br />Primary antibody: <a href="/products/HA751506" style="font-weight: bold;text-decoration: underline;">HA751506</a>, 1/1,000, 1 hour at room temperature.<br />Secondary antibody: <a href="/products/HA1119" style="font-weight: bold;text-decoration: underline;">HA1119</a>, 20 minutes at room temperature.

    Application: Immunohistochemistry (IHC-P)

    Species: Mouse
    Tissue: Alveolar bone
    Sample: Paraffin-embedded section

    Antigen retrieval: Heat-mediated, Tris-EDTA buffer (pH 9.0), 20 minutes at 95℃.

    Wash buffer: 1× TBST
    Endogenous peroxidase blocking: 3% H₂O₂, 10 minutes at room temperature.
    Blocking: 1% BSA + 10% normal goat serum, 10 minutes at room temperature.
    Primary antibody: HA751506, 1/1,000, 1 hour at room temperature.
    Secondary antibody: HA1119, 20 minutes at room temperature.

  • Application: IHC-Fr<br /><br />Species: Mouse<br /><br />Site: kidney<br /><br />Sample: Frozen section<br /><br />Antibody concentration: 1/500<br /><br />Antigen retrieval: Not required

    Application: IHC-Fr

    Species: Mouse

    Site: kidney

    Sample: Frozen section

    Antibody concentration: 1/500

    Antigen retrieval: Not required

  • Application: IHC-Fr<br /><br />Species: Rat<br /><br />Site: kidney<br /><br />Sample: Frozen section<br /><br />Antibody concentration: 1/500<br /><br />Antigen retrieval: Not required

    Application: IHC-Fr

    Species: Rat

    Site: kidney

    Sample: Frozen section

    Antibody concentration: 1/500

    Antigen retrieval: Not required

  • Western blot analysis of Osteopontin/SPP1 on different lysates with Rabbit anti-Osteopontin/SPP1 antibody (<a href="/products/HA751506" style="font-weight: bold;text-decoration: underline;">HA751506</a>) at 1/5,000 dilution.<br /><br />Lane 1: RAW264.7 cell lysate<br />Lane 2: RAW264.7 treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours cell lysate<br />Lane 3: RAW264.7 cell lysate<br />Lane 4: RAW264.7 add 100nM TPA overnight, then treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours cell lysate<br /><br />Lysates/proteins at 20 µg/Lane.<br /><br />Predicted band size: 32 kDa<br />Observed band size: 60 kDa<br /><br />Exposure time: Lane 1-2: 46 seconds; Lane 3-4: 4 seconds; ECL: K1801;<br />4-20% SDS-PAGE gel.<br /><br />Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (<a href="/products/HA751506" style="font-weight: bold;text-decoration: underline;">HA751506</a>) at 1/5,000 dilution was used in primary antibody dilution (<a href="/products/K1803" style="font-weight: bold;text-decoration: underline;">K1803</a>) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (<a href="/products/HA1001" style="font-weight: bold;text-decoration: underline;">HA1001</a>) at 1/50,000 dilution was used for 1 hour at room temperature.

    Western blot analysis of Osteopontin/SPP1 on different lysates with Rabbit anti-Osteopontin/SPP1 antibody (HA751506) at 1/5,000 dilution.

    Lane 1: RAW264.7 cell lysate
    Lane 2: RAW264.7 treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours cell lysate
    Lane 3: RAW264.7 cell lysate
    Lane 4: RAW264.7 add 100nM TPA overnight, then treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours cell lysate

    Lysates/proteins at 20 µg/Lane.

    Predicted band size: 32 kDa
    Observed band size: 60 kDa

    Exposure time: Lane 1-2: 46 seconds; Lane 3-4: 4 seconds; ECL: K1801;
    4-20% SDS-PAGE gel.

    Proteins were transferred to a PVDF membrane and blocked with 5% NFDM/TBST for 1 hour at room temperature. The primary antibody (HA751506) at 1/5,000 dilution was used in primary antibody dilution (K1803) at 4℃ overnight. Goat Anti-Rabbit IgG - HRP Secondary Antibody (HA1001) at 1/50,000 dilution was used for 1 hour at room temperature.

  • Immunocytochemistry analysis of RAW264.7 cells untreated / treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours labeling Osteopontin/SPP1 with Rabbit anti-Osteopontin/SPP1 antibody (<a href="/products/HA751506" style="font-weight: bold;text-decoration: underline;">HA751506</a>) at 1/100 dilution.<br /><br />Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Osteopontin/SPP1 antibody (<a href="/products/HA751506" style="font-weight: bold;text-decoration: underline;">HA751506</a>) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor&trade; 488, <a href="/products/HA1121" style="font-weight: bold;text-decoration: underline;">HA1121</a>) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.<br /><br />Beta tubulin (<a href="/products/HA601187" style="font-weight: bold;text-decoration: underline;">HA601187</a>, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor&trade; 594, <a href="/products/HA1126" style="font-weight: bold;text-decoration: underline;">HA1126</a>) was used as the secondary antibody at 1/1,000 dilution.

    Immunocytochemistry analysis of RAW264.7 cells untreated / treated with 100ng/mL LPS for 4 hours, add 1μg/mL BFA for last 3 hours labeling Osteopontin/SPP1 with Rabbit anti-Osteopontin/SPP1 antibody (HA751506) at 1/100 dilution.

    Cells were fixed in 4% paraformaldehyde for 15 minutes at room temperature, permeabilized with 0.1% Triton X-100 in PBS for 15 minutes at room temperature, then blocked with 1% BSA in 10% negative goat serum for 1 hour at room temperature. Cells were then incubated with Rabbit anti-Osteopontin/SPP1 antibody (HA751506) at 1/100 dilution in 1% BSA in PBST overnight at 4 ℃. Goat Anti-Rabbit IgG H&L (iFluor™ 488, HA1121) was used as the secondary antibody at 1/1,000 dilution. PBS instead of the primary antibody was used as the secondary antibody only control. Nuclear DNA was labelled in blue with DAPI.

    Beta tubulin (HA601187, red) was stained at 1/100 dilution overnight at +4℃. Goat Anti-Mouse IgG H&L (iFluor™ 594, HA1126) was used as the secondary antibody at 1/1,000 dilution.

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