Anti-SPARC antibody [EPR25122-122]

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SPARC antibody [EPR25122-122] (AB290636)

Immunohistochemical analysis of paraffin-embedded mouse hepatocellular tissue labelling SPARC with ab290636 at 1/15000 (0.041 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) . Positive staining on interstitial cells of mouse hepatocellular carcinoma. The section was incubated with ab290636 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.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SPARC antibody [EPR25122-122] (AB290636)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Rat primary neuron cells labelling SPARC with ab290636 at 1/600 dilution (0.1μg) (Right) compared with a Rabbit monoclonal IgG (ab172730) (Left). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SPARC antibody [EPR25122-122] (AB290636)

Immunohistochemical analysis of paraffin-embedded Mouse kidney tissue labelling SPARC with ab290636 at 1/15000 (0.041 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) . Positive staining on glomerulus and endothelium of mouse kidney. The section was incubated with ab290636 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-SPARC antibody [EPR25122-122] (AB290636)

Immunohistochemical analysis of paraffin-embedded mouse cerebrum tissue labelling SPARC with ab290636 at 1/15000 (0.041 μg/ml) followed by a ready to use LeicaDS9800 (BOND™ Polymer Refine Detection) . Positive staining on microglia of mouse cerebrum. The section was incubated with ab290636 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.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SPARC antibody [EPR25122-122] (AB290636)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized bEnd.3 (mouse brain endothelioma cell line) cells labelling SPARC with primary antibody anti-SPARC (ab290636) at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) secondary antibody at 1/1000 dilution (2.0 µg/mL). Confocal image showing cytoplasmic staining in bEnd.3 cells. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL). The nuclear counter stain is DAPI (blue). The secondary antibody only control is : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2.0 µg/mL).

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-SPARC antibody [EPR25122-122] (AB290636)

Immunofluorescent analysis of 4% paraformaldehyde-fixed, 0.1% Triton X-100 permeabilized C6 (rat glial tumour cell line) cells labelling SPARC with primary antibody anti-SPARC (ab290636) at 1/100 dilution, followed by Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) (ab150081) secondary antibody at 1/1000 dilution (2.0 µg/mL). Confocal image showing cytoplasmic staining in C6 cells. Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) (ab195889) was used to counterstain tubulin at 1/200 dilution (2.5 µg/mL). The nuclear counter stain is DAPI (blue). The secondary antibody only control is : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) at 1/1000 dilution (2.0 µg/mL).

• Flow Cyt (Intra)

Supplier Data

Flow Cytometry (Intracellular) - Anti-SPARC antibody [EPR25122-122] (AB290636)

Intracellular flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Mouse primary neuron cell cells labelling SPARC with ab290636 at 1/600 dilution (0.1μg) (Right) compared with a Rabbit monoclonal IgG (ab172730) (Left) isotype control 0. A Goat Anti-Rabbit IgG (Alexa Fluor^® 488, ab150081) at 1/2000 dilution was used as the secondary antibody.

• IP

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Immunoprecipitation - Anti-SPARC antibody [EPR25122-122] (AB290636)

SPARC was immunoprecipitated from rat brain tissue lysate with ab290636 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab290636 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Rat brain tissue lysate 10 μg

Lane 2 : ab290636 IP in Rat brain tissue lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab290636 in rat brain tissue lysate

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

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-SPARC antibody [EPR25122-122] (ab290636)

Predicted band size: 35 kDa

false

• IP

Supplier Data

Immunoprecipitation - Anti-SPARC antibody [EPR25122-122] (AB290636)

SPARC was immunoprecipitated from Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate with ab290636 at 1/30 dilution (2μg in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab290636 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution.

Lane 1 : Neuro-2a (mouse neuroblastoma neuroblast) whole cell lysate 10 μg

Lane 2 : ab290636 IP in Neuro-2a whole cell lysate

Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab290636 in Neuro-2a whole cell lysate

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

Exposure time : 10 seconds

All lanes:

Immunoprecipitation - Anti-SPARC antibody [EPR25122-122] (ab290636)

Predicted band size: 35 kDa

false

• WB

Supplier Data

Western blot - Anti-SPARC antibody [EPR25122-122] (AB290636)

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

The expression profile/molecular weight observed is consistent with that described in the literature (PMID : 29449802; PMID : 9008236).
SPARC is a glycosylated protein and can be deglycosylated by Protein Deglycosylation MIX II.
The additional band is expected to be a SPARC cleavage product (PMID : 9008236).

All lanes:

Western blot - Anti-SPARC antibody [EPR25122-122] (ab290636) at 1/1000 dilution

Lane 1:

Untreated mouse brain tissue lysate at 15 µg

Lane 2:

Mouse brain tissue lysate treated with Protein Deglycosylation MIX II at 15 µg

Lane 3:

Untreated rat brain tissue lysate at 15 µg

Lane 4:

Rat brain tissue lysate treated with Protein Deglycosylation MIX II at 15 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: 37 kDa,40 kDa

false

Exposure time: 103s

• WB

Supplier Data

Western blot - Anti-SPARC antibody [EPR25122-122] (AB290636)

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

The expression profile/molecular weight observed is consistent with that described in the literature (PMID : 29449802; PMID : 9008236).
The additional band is expected to be a SPARC cleavage product (PMID : 9008236).

Exposure time :

Lane 2 : 5.5 seconds
Lane 1, 3-6 : 3.25 seconds

All lanes:

Western blot - Anti-SPARC antibody [EPR25122-122] (ab290636) at 1/1000 dilution

Lane 1:

Mouse brain tissue lysate at 20 µg

Lane 2:

Mouse heart tissue lysate at 20 µg

Lane 3:

Mouse placenta tissue lysate at 20 µg

Lane 4:

Rat brain tissue lysate at 20 µg

Lane 5:

Rat heart tissue lysate at 20 µg

Lane 6:

Rat placenta tissue 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 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 37 kDa,40 kDa

false

• WB

Supplier Data

Western blot - Anti-SPARC antibody [EPR25122-122] (AB290636)

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

Exposure time : 3.25 seconds.

The expression profile/molecular weight observed is consistent with that described in the literature (PMID : 29449802; PMID : 9008236).
The additional band is expected to be a SPARC cleavage product (PMID : 9008236).

All lanes:

Western blot - Anti-SPARC antibody [EPR25122-122] (ab290636) at 1/1000 dilution

Lane 1:

C2C12 (mouse myoblast) whole cell lysate at 20 µg

Lane 2:

bEnd.3 (mouse brain endothelioma) whole cell lysate at 20 µg

Lane 3:

Neuro-2a (mouse neuroblastoma) whole cell lysate at 20 µg

Lane 4:

C6 (rat glioma) 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 1/100000 dilution

Predicted band size: 35 kDa

Observed band size: 37 kDa,40 kDa

false