Anti-Osteopontin antibody [EPR21139-316]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Immunohistochemical analysis of paraffin-embedded human kidney tissue labeling Osteopontin with ab214050 at 1/2000 dilution, followed by Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) Ready to use. Cytoplasmic staining in distal tubules of human kidney (PMID : 17938278), performed on a Leica Biosystems BOND® RX instrument. Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat-mediated antigen retrieval with sodium citrate buffer (pH 6.0, epitope retrieval solution 1) for 10 minutes.

• WB

Supplier Data

Western blot - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Blocking and diluting buffer concentration : 5% NFDM/TBST.

We suggest treating cells with BFA which inhibits protein secretion.

All lanes:

Western blot - Anti-Osteopontin antibody [EPR21139-316] (ab214050) at 1/1000 dilution

Lane 1:

Untreated HepG2 (Human hepatocellular carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

HepG2 treated with 300ng/ml BFA for 3h whole cell lysate at 20 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 60 kDa

false

Exposure time: 60s

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Immunohistochemical analysis of paraffin-embedded human liver tissue labeling Osteopontin with ab214050 at 1/2000 dilution, followed by Goat Anti-Rabbit IgG H&L (HRP) Ready to use. Cytoplasmic staining in bile ducts of human liver (PMID : 16221502). Counter stained with hematoxylin.

Secondary antibody only control : Used PBS instead of primary antibody, secondary antibody is Goat Anti-Rabbit IgG H&L (HRP) Ready to use.

Perform heat-mediated antigen retrieval using ab93684 (Tris/EDTA buffer, pH 9.0).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized U-87 MG cell line labelling Osteopontin with ab214050 at 1/50 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution (green). Confocal image showing cytoplamic staining in U-87 MG cell line. The nuclear counter stain is DAPI (blue).

The negative controls is as follows :
-ve control : Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) at 1/200 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150077) secondary antibody at 1/1000 dilution.

• IP

Supplier Data

Immunoprecipitation - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Osteopontin was immunoprecipitated from 0.35 mg of human kidney lysate with ab214050 at 1/30 dilution. Western blot was performed from the immunoprecipitate using ab214050 at 1/1000 dilution. VeriBlot for IP Detection Reagent (HRP) (ab131366), was used for detection at 1/5,000 dilution

Lane 1 : Human kidney lysate 10 μg (Input).

Lane 2 : ab214050 IP in human kidney lysate.

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab214050 in human kidney lysate.

Blocking and dilution buffer and concentration : 5% NFDM/TBST.

Exposure time : 3 minutes.

The multiple bands observed are likely due to expression of different isoforms, as well as glycosylation (PMID : 8761944; PMID : 11375993; PMID : 8535240; PMID : 12890718).

All lanes:

Immunoprecipitation - Anti-Osteopontin antibody [EPR21139-316] (ab214050)

Predicted band size: 35 kDa

false

• WB

Supplier Data

Western blot - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Blocking/Dilution buffer : 5% NFDM/TBST.

The multiple bands observed are likely due to expression of different isoforms, as well as glycosylation (PMID : 8761944; PMID : 11375993; PMID : 8535240; PMID : 12890718).

The identity of the 90kDa band is unknown.

All lanes:

Western blot - Anti-Osteopontin antibody [EPR21139-316] (ab214050) at 1/1000 dilution

Lane 1:

Human urine at 20 µg

Lane 2:

Human fetal kidney lysate at 10 µg

Secondary

Lane 1:

Western blot - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/1000 dilution

Lane 2:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 45 kDa,60 kDa,70 kDa

false

Exposure time: 26s

• WB

Supplier Data

Western blot - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Blocking/Dilution buffer : 5% NFDM/TBST.

The his-tagged recombinant protein was produced in a mammalian expression system, thereby allowing it to be post-translationally modified.

All lanes:

Western blot - Anti-Osteopontin antibody [EPR21139-316] (ab214050) at 1/1000 dilution

All lanes:

Human Osteopontin recombinant protein (aa17-300) produced in a mammalian expression at 0.01 µg

Secondary

All lanes:

Western blot - Goat Anti-Rabbit IgG H&L (HRP) (<a href='/en-us/products/secondary-antibodies/goat-rabbit-igg-h-l-hrp-ab97051'>ab97051</a>) at 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 60-65 kDa

false

Exposure time: 26s

• WB

CiteAb

Western blot - Anti-Osteopontin antibody [EPR21139-316] (AB214050)

Osteopontin western blot using anti-Osteopontin antibody [EPR21139-316] ab214050. Publication image and figure legend from Chen, P., Chen, Y., et al., 2019, Aging (Albany NY), PubMed 31449494.

ab214050 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab214050 please see the product overview.

Hub proteins and potential pathway proteins were increased in AS plaques, especially in tissue sections of ruptured plaques. (A) Detection of hub proteins (SPP1, MMP9, CCL4, and CCL18) and potential pathway proteins (PPARγ, C/EBP and FABP4) in ruptured plaques and adjacent normal tissues by western blots. β-actin was used as a loading control. Bands were quantified with ImageJ software. (B) Line chart showing IOD/area of proteins in immunoblot analysis. Red squares represent AS plaques, and the green circles indicate adjacent normal tissues. The different colored lines represent the trend of protein expression. (C, D) H&E (a) and Movat (b and c, low and high power) staining and immunoperoxidase antibody staining using anti-CD68 (d), anti-SPP1 (e), anti-MMP9 (f), anti-CCL4 (g), and anti-CCL18 (h). Ca, calcification; PR, plaque rupture; Th, thrombus.

false