Anti-SPIB antibody [EPR26784-117]

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SPIB antibody [EPR26784-117] (AB309346)

Immunohistochemical analysis of paraffin-embedded Human Non-Hodgkin ly tissue labeling SPIB with ab309346 at 1/100 (5.31 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human Non-Hodgkin lymphoma (PMID : 23165482).The section was incubated with ab309346 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SPIB antibody [EPR26784-117] (AB309346)

Immunohistochemical analysis of paraffin-embedded Human tonsil tissue labeling SPIB with ab309346 at 1/100 (5.31 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on human tonsil (PMID : 23165482).The section was incubated with ab309346 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SPIB antibody [EPR26784-117] (AB309346)

Immunohistochemical analysis of paraffin-embedded Human colon tissue labeling SPIB with ab309346 at 1/100 (5.31 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Nuclear staining on immune cells in human colon (PMID : 23360902).The section was incubated with ab309346 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SPIB antibody [EPR26784-117] (AB309346)

Immunofluorescent analysis of 4% Paraformaldehyde-fixed, 0.1% TritonX-100 permeabilized Raji (human Burkitt's lymphoma B lymphocyte) cells labelling SPIB with ab309346 at 1/50 (10.62 ug/ml) dilution, followed by ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed antibody at 1/1000 2ug/ml dilution (Green). Confocal image showing nuclear staining in Raji cell line.Negative control : Jurkat (PMID : 8691135).Image was taken with a confocal microscope(Leica-Microsystems, TCS SP8). is observed. ab195889 Anti-alpha Tubulin mouse monoclonal antibody - Microtubule Marker (Alexa Fluor® 594) was used to counterstain tubulin at 1/200 2.5ug/ml dilution (Red). The Nuclear counterstain was DAPI (Blue). Secondary antibody only control : Secondary antibody is ab150081 Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed at 1/1000 2ug/ml dilution.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SPIB antibody [EPR26784-117] (AB309346)

Flow cytometric analysis of 4% paraformaldehyde fixed 90% methanol permeabilized Jurkat (human T cell leukemia T lymphocyte from peripheral blood, Left) / Raji (human Burkitt's lymphoma B lymphocyte, Right) cells labelling SPIB with ab309346 at 1/50 dilution (1 ug)/Red (Red) compared with a Rabbit monoclonal IgG (ab172730) (Black) isotype control and an unlabelled control (cells without incubation with primary antibody and secondary antibody) (Blue). A Goat Anti-Rabbit IgG (Alexa Fluor® 488, ab150081) at 1/5000 dilution was used as the secondary antibody. Negative control : Jurkat (PMID : 8691135, 11841448).

• IP

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Immunoprecipitation - Anti-SPIB antibody [EPR26784-117] (AB309346)

SPIB was immunoprecipitated from 0.35 mg Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate with ab309346 at 1/30 dilution (2ug in 0.35mg lysates). Western blot was performed on the immunoprecipitate using ab309346 at 1/1000 dilution. VeriBlot for IP secondary antibody(HRP)(ab131366) was used at 1/5000 dilution. Lane 1 : Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate Lane 2 : ab309346 IP in Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate Lane 3 : Rabbit monoclonal IgG (ab172730) instead of ab309346 in Raji whole cell lysate Blocking and dilution buffer and concentration : 5% NFDM/TBST. Exposure time : 180 seconds

All lanes:

Immunoprecipitation - Anti-SPIB antibody [EPR26784-117] (ab309346) at 1/30 dilution

All lanes:

Raji (human Burkitt's lymphoma B lymphocyte) whole cell lysate

Secondary

All lanes:

Immunoprecipitation - VeriBlot for IP Detection Reagent (HRP) (<a href='/en-us/products/reagents/veriblot-for-ip-detection-reagent-hrp-ab131366'>ab131366</a>) at 1/5000 dilution

false

Exposure time: 180s

• WB

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Western blot - Anti-SPIB antibody [EPR26784-117] (AB309346)

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

Negative control : Jurkat (PMID : 8691135, 11841448), HEK-293.

The expression profile/ molecular weight observed is consistent with what has been described in the literature (PMID : 8691135).

In lanes 1-5, the lysates were stored at -80℃ prior to Western Blotting. The bands beneath the target band (46 kDa) are likely to be degradation products. In lanes 6-7, the lysates were freshly made and used for Western Blotting immediately to minimize protein degradation.

This blot was developed using a high sensitivity ECL substrate. The high-sensitivity ECL substrate used allows for the detection of proteins in the mid-femtogram range.

In Western blot, anti- H3 antibody (ab176842) loading control staining at 1/100000 dilution.

Exposure time :

Lanes 1-5 : 48 seconds

Lanes 6-7 : 26 seconds

All lanes:

Western blot - Anti-SPIB antibody [EPR26784-117] (ab309346) at 1/1000 dilution

Lane 1:

Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 2:

HDLM-2 (human Hodgkin lymphoma) whole cell lysate at 20 µg

Lane 3:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 4:

Jurkat (human T cell leukemia T lymphocyte) whole cell lysate at 20 µg

Lane 5:

HEK-293 (human embryonic kidney epithelial cell) whole cell lysate at 20 µg

Lane 6:

Raji whole cell lysate at 20 µg

Lane 7:

Jurkat 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

Observed band size: 46 kDa

true

• WB

Lab

Western blot - Anti-SPIB antibody [EPR26784-117] (AB309346)

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000 dilution.

We recommend optimizing the protocol to enhance signal intensity by increasing protein loading (30-50 μg/lane), reducing primary antibody dilution (1 : 500), and utilizing femtogram-level ECL substrates.

All lanes:

Western blot - Anti-SPIB antibody [EPR26784-117] (ab309346) at 1/1000 dilution

Lane 1:

Raji (human Burkitts lymphoma B lymphocyte) whole cell lysate at 20 µg

Lane 2:

Daudi (human Burkitts lymphoma lymphoblast) whole cell lysate at 20 µg

Lane 3:

Human colon tissue lysate at 20 µg

Lane 4:

Human tonsil tissue lysate at 20 µg

Lane 5:

Human spleen 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 1/20000 dilution

Observed band size: 46 kDa

false

Exposure time: 180s

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-SPIB antibody [EPR26784-117] (AB309346)

Immunohistochemical analysis of paraffin-embedded Human liver tissue labeling SPIB with ab309346 at 1/100 (5.31 ug/ml) followed by a ready to use LeicaDS9800 (Bond™ Polymer Refine Detection). Negative control : No staining on human liver (PMID : 26610895).The section was incubated with ab309346 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 was performed with Tris-EDTA buffer (pH 9.0, Epitope Retrieval Solution2) for 20 mins