Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free (AB236213)

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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214050).

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free (AB236213)

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.

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214050).

• IP

Supplier Data

Immunoprecipitation - Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free (AB236213)

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 1/5000 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).

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214050).

All lanes:

Immunoprecipitation - Anti-Osteopontin antibody [EPR21139-316] (<a href='/ko/products/primary-antibodies/osteopontin-antibody-epr21139-316-ab214050'>ab214050</a>)

Predicted band size: 35 kDa

false

• WB

Lab

Western blot - Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free (AB236213)

This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (ab214050).

Western blot : Anti-Osteopontin antibody [EPR21139-316] ab214050 staining at 1/1000 dilution, shown in green; Mouse anti-CANX ab238078 loading control staining at 1/20000 dilution, shown in magenta. A band was observed at 51 kDa. To generate this image, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5% Milk in TBS-0.1 % Tween^® 20 (TBS-T) before incubation with primary antibodies overnight at 4 °C. Blots were washed four times in TBS-T, incubated with secondary antibodies for 1 h at room temperature, washed again four times then imaged. Secondary antibodies used were Goat anti-Rabbit 800CW and Goat anti-Mouse 680RD at 1/20,000 dilution.

All lanes:

Western blot - Anti-Osteopontin antibody [EPR21139-316] (<a href='/ko/products/primary-antibodies/osteopontin-antibody-epr21139-316-ab214050'>ab214050</a>) at 1/1000 dilution

Lane 1:

Wild-type A549 BFA (5 ug/mL, 6h) cell lysates at 20 µg

Lane 2:

Western blot - Human SPP1 knockout A549 cell line (<a href='/ko/products/cell-lines/human-spp1-knockout-a549-cell-line-ab287778'>ab287778</a>) at 20 µg

Lane 3:

SPP1 knockout A549 <a href='/ko/products/cell-lines/human-spp1-knockout-a549-cell-line-ab287778'>ab287778</a> BFA (5 ug/mL, 6h) cell lysates at 20 µg

Lane 4:

SPP1 knockout A549 <a href='/ko/products/cell-lines/human-spp1-knockout-a549-cell-line-ab287778'>ab287778</a> BFA (0 ug/mL, 6h) cell lysates at 20 µg

Lane 5:

HepG2 BFA (5 ug/mL, 6h) cell lysates at 20 µg

Lane 6:

HepG2 BFA (0 ug/mL, 6h) cell lysates at 20 µg

Secondary

All lanes:

Goat anti-Rabbit 800CW & Goat anti-Mouse 680RD at 1/20000 dilution

Predicted band size: 35 kDa

Observed band size: 51 kDa

false

• WB

Supplier Data

Western blot - Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free (AB236213)

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.

This data was developed using ab214050, the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide.

All lanes:

Western blot - Anti-Osteopontin antibody [EPR21139-316] (<a href='/ko/products/primary-antibodies/osteopontin-antibody-epr21139-316-ab214050'>ab214050</a>) 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='/ko/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='/ko/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

Lab

Western blot - Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free (AB236213)

This data was developed using ab214050, the same antibody clone in a different buffer formulation.

In Western blot, anti-GAPDH antibody (ab181602) loading control staining at 1/1000000 dilution.

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

Low expressing sample : MCF7 (PMID : 16288209); PANC-1 (PMID : 34572536).

All lanes:

Western blot - Anti-Osteopontin antibody [EPR21139-316] (<a href='/ko/products/primary-antibodies/osteopontin-antibody-epr21139-316-ab214050'>ab214050</a>) at 1/1000 dilution

Lane 1:

A549 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 2:

NCI-H460 (human Large Cell Lung Cancer epithelial cell) whole cell lysate at 20 µg

Lanes 3 - 4:

NCI-H1299 (human lung carcinoma epithelial cell) whole cell lysate at 20 µg

Lane 5:

MCF7 (human breast adenocarcinoma epithelial cell) whole cell lysate at 20 µg

Lane 6:

PANC-1 (human pancreatic epithelioid carcinoma epithelial cell) whole cell lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 36 kDa

false

Exposure time: 80s

• WB

Supplier Data

Western blot - Anti-Osteopontin antibody [EPR21139-316] - BSA and Azide free (AB236213)

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

We suggest treating cells with BFA which inhibits protein secretion.

This data was developed using ab214050, the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide.

All lanes:

Western blot - Anti-Osteopontin antibody [EPR21139-316] (<a href='/ko/products/primary-antibodies/osteopontin-antibody-epr21139-316-ab214050'>ab214050</a>) 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='/ko/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