Anti-Osteopontin antibody [RM1018] - BSA and Azide free

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human liver tissue labelling Osteopontin with ab283656 at 1/2000 (0.232 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the bile ducts in human liver. The section was incubated with ab283656 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Human kidney tissue labelling Osteopontin with ab283656 at 1/2000 (0.232 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on distal tubules in human kidney. The section was incubated with ab283656 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

Supplier Data

Immunoprecipitation - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

Osteopontin was immunoprecipitated from 0.35 mg Human kidney lysate 10ug with ab283656 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283656 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Human kidney lysate 10ug

Lane 2 : ab283656 IP in Human kidney lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283656 in Human kidney lysate

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

Exposure time : 1 min.

All lanes:

Immunoprecipitation - Anti-Osteopontin antibody [RM1018] (<a href='/en-us/products/primary-antibodies/osteopontin-antibody-rm1018-ab283656'>ab283656</a>)

Predicted band size: 35 kDa

Observed band size: 60 kDa

false

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

Immunohistochemical analysis of paraffin-embedded Mouse liver tissue labelling Osteopontin with ab283656 at 1/2000 (0.232 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Positive staining on the bile ducts in mouse liver. The section was incubated with ab283656 for 30 mins at room temperature. The immunostaining was performed on a Leica Biosystems BOND® RX instrument Counterstained with Hematoxylin.

Secondary antibody only control : Secondary antibody is a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection).

Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins.

• IP

Supplier Data

Immunoprecipitation - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

Osteopontin was immunoprecipitated from 0.35 mg RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 3h then add 300ng/ml BFA for another 3h whole cell lysate 10ug with ab283656 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab283656 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) treated with 100ng/ml LPS for 3h then add 300ng/ml BFA for another 3h whole cell lysate 10ug

Lane 2 : ab283656 IP in RAW 264.7 treated with 100ng/ml LPS for 3h then add 300ng/ml BFA for another 3h whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab283656 in RAW 264.7 treated with 100ng/ml LPS for 3h then add 300ng/ml BFA for another 3h whole cell lysate

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

Exposure time : 32 seconds.

Lower bands should be MMP-cleaved Osteopontin. (PMID : 11375993)

All lanes:

Immunoprecipitation - Anti-Osteopontin antibody [RM1018] (<a href='/en-us/products/primary-antibodies/osteopontin-antibody-rm1018-ab283656'>ab283656</a>)

Predicted band size: 35 kDa

Observed band size: 60 kDa

false

• WB

Lab

Western blot - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation. Blocking and diluting buffer and concentration : 5% NFDM/TBST. ab181602 was used as a GAPDH loading control. This antibody is not recommended for use in WB with mouse tissue samples. We suggest treating cells with BFA which inhibits protein secretion.

All lanes:

Western blot - Anti-Osteopontin antibody [RM1018] (<a href='/en-us/products/primary-antibodies/osteopontin-antibody-rm1018-ab283656'>ab283656</a>) at 1/1000 dilution

Lane 1:

Mouse kidney tissue lysate at 20 µg

Lane 2:

Mouse placenta tissue lysate at 20 µg

Lane 3:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 4:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 4h whole cell lysate at 20 µg

Lane 5:

RAW 264.7 treated with 1000ng/ml BFA for 3h whole cell lysate at 20 µg

Lane 6:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 4h then add 1000ng/ml BFA for another 3h whole cell lysate at 20 µg

Lane 7:

Human kidney tissue lysate at 20 µg

Lane 8:

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

Secondary

All lanes:

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

Predicted band size: 35 kDa

Observed band size: 60 kDa

false

Exposure time: 180s

• WB

Lab

Western blot - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

Blocking and diluting buffer and concentration : 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).

Exposure time : 2 min.

All lanes:

Western blot - Anti-Osteopontin antibody [RM1018] (<a href='/en-us/products/primary-antibodies/osteopontin-antibody-rm1018-ab283656'>ab283656</a>) at 1/1000 dilution

Lane 1:

Human kidney lysate at 20 µg

Lane 2:

MDA-MB-435S (Human mammary gland ductal carcinoma melanocyte) whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<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 kDa

false

• WB

Lab

Western blot - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

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

Exposure time : 37 seconds.

All lanes:

Western blot - Anti-Osteopontin antibody [RM1018] (<a href='/en-us/products/primary-antibodies/osteopontin-antibody-rm1018-ab283656'>ab283656</a>) at 1/1000 dilution

Lane 1:

RAW 264.7 (Mouse Abelson murine leukemia virus-induced tumor macrophage) whole cell lysate at 20 µg

Lane 2:

RAW 264.7 treated with 100ng/ml lipopolysaccharide (LPS) for 3h then add 300ng/ml BFA for another 3h whole cell lysate at 20 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG, (H+L), Peroxidase conjugated (<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 kDa

false

• cELISA

Supplier Data

Competitive ELISA - Anti-Osteopontin antibody [RM1018] - BSA and Azide free (AB283669)

This data was developed using ab283656, the same antibody clone in a different buffer formulation.

ELISA using ab283656 at varying antibody concentrations and antigen concentration at 1000 ng/ml. An Alkaline Phosphatase-conjugated AffiniPure Goat Anti-Rabbit IgG (H+L) (1/2500) was used as the secondary antibody.