Anti-Semaphorin 4D/CD100 antibody [EP3569]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

Immunocytochemistry/ Immunofluorescence analysis of Jurkat (Human T cell leukemia cell line from peripheral blood) cells labeling Semaphorin 4D/CD100 with Purified ab134128 at 1 : 250 dilution (4.8μg/ml). Cells were fixed in 4% Paraformaldehyde and permeabilized with 0.1% tritonX-100. Cells were counterstained with ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) 1 : 200 (2.5 μg/ml). ab150077 Goat anti rabbit IgG(Alexa Fluor^® 488) was used as the secondary antibody at 1 : 1000 dilution. DAPI nuclear counterstain. PBS instead of the primary antibody was used as the secondary antibody only control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

Immunohistochemical analysis of paraffin embedded Human tonsil tissue labelling Semaphorin 4D/CD100 with ab134128 at 1/100 dilution

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of Human tonsil tissue sections labeling Semaphorin 4D/CD100 with Purified ab134128 at 1 : 120 dilution (10 μg/ml). Heat mediated antigen retrieval was performed using EDTA Buffer PH9. Tissue was counterstained with Hematoxylin. ImmunoHistoProbe one step HRP Polymer (ready to use) secondary antibody was used at 1 : 0 dilution. PBS instead of the primary antibody was used as the negative control.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

Immunohistochemical analysis of paraffin embedded Human colon tissue labelling Semaphorin 4D/CD100 using ab134128 at 1/100 dilution. Positive staining of lymphocytes inclusion.

• WB

Unknown

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

HRP labelled goat anti rabbit secondary antibody used at 1/2000 dilution

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (ab134128) at 1/1000 dilution

Lane 1:

Camptothecin treated Jurkat cell lysate at 10 µg

Lane 2:

Jurkat cell lysate at 10 µg

Lane 3:

Human spleen lysate at 10 µg

Lane 4:

Human fetal kidney lysate at 10 µg

Predicted band size: 96 kDa

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• WB

Lab

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

Western blot : Anti-SEMA4D antibody [EP3569] (ab134128) staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab134128 was shown to bind specifically to SEMA4D. A band was observed at 90-120 kDa in wild-type HCT 116 cell lysates with no signal observed at this size in SEMA4D knockout cell line. To generate this image, wild-type and SEMA4D knockout HCT 116 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (ab134128) at 1/2000 dilution

Lane 1:

Wild-type HCT 116 cell lysate at 20 µg

Lane 2:

Western blot - Human SEMA4D knockout HCT116 cell line (<a href='/en-us/products/cell-lines/human-sema4d-knockout-hct116-cell-line-ab289144'>ab289144</a>)

Lane 2:

SEMA4D knockout HCT 116 cell lysate at 20 µg

Lane 3:

HDLM-2 UNBOILED cell lysate at 20 µg

Lane 4:

PC-3 UNBOILED cell lysate at 20 µg

Secondary

Lanes 1 - 4:

Goat anti-Rabbit IgG H&L 800CW at 1/20000 dilution

Lanes 1 - 4:

Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

Observed band size: 90-120 kDa

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• WB

Lab

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

Western blot : Anti-SEMA4D antibody [EP3569] (ab134128) staining at 1/2000 dilution, shown in green; Mouse anti-Alpha Tubulin [DM1A] (ab7291) loading control staining at 1/20000 dilution, shown in magenta. In Western blot, ab134128 was shown to bind specifically to SEMA4D. A band was observed at 95-100 kDa in wild-type A549 cell lysates with no signal observed at this size in SEMA4D knockout cell line. To generate this image, wild-type and SEMA4D knockout A549 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 3 % 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 IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution.

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (ab134128) at 1/2000 dilution

Lane 1:

Wild-type A549 cell lysate at 40 µg

Lane 2:

SEMA4D knockout A549 cell lysate at 40 µg

Lane 3:

HDLM-2 cell lysate at 20 µg

Lane 4:

PC-3 cell lysate at 20 µg

Secondary

All lanes:

Goat anti-Rabbit IgG H&L 800CW and Goat anti-Mouse IgG H&L 680RD at 1/20000 dilution

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• WB

Unknown

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (AB134128)

Blocking and diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Semaphorin 4D/CD100 antibody [EP3569] (ab134128) at 0.2 µg/mL

All lanes:

SH-SY5Y (Human neuroblastoma epithelial cell) 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: 96 kDa

Observed band size: 120 kDa,150 kDa

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