Anti-SLP76 (phospho Y145) 抗体 [EP2853Y]

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (AB75829)

ab75829 staining SLP76 in Jurkat by Immunocytochemistry. Tissue was fixed with 4% paraformaldehyde. Samples were incubated with primary antibody (4.5 μg/ml). ab150077 AlexaFluor®488 Goat anti-Rabbit (1/1000) was used as the secondary antibody. ab195889 Anti-alpha Tubulin antibody [DM1A] - Microtubule Marker (Alexa Fluor^® 594) at 2.5 μg/ml and DAPI were used as counter stains.

Confocal image showing the expression was increased after treatment with pervanadate (1mM 30min) and then decreased after treatment with the Lambda Protein Phosphatase 31°C for 2h

• ICC/IF

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Immunocytochemistry/ Immunofluorescence - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (AB75829)

ab75829 staining SLP76 (phospho Y145) antibody in both treated (50mM of pervanadate for 5 minutes) and untreated Jurkat (human acute T cell leukemia) cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with 4% Paraformaldehyde and permeabilized with 0.1% Triton X-100. Samples were incubated with primary antibody at a dilution of 1/500. A goat anti rabbit IgG (Alexa Fluor^® 488) (ab150077) was used as the secondary antibody at a dilution of 1/1000.

DAPI was used as a nuclear counterstain.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (AB75829)

Intracellular flow cytometric analysis of permeabilized Jurkat cells, untreated (green) or pervanadate treated (red) using ab75829 at a 1/100 dilution. Also, pervanadate Jurkat cells using the same antibody preincubated with phospho SLP76 peptide (blue) or non phospho SLP76 peptide (orange).

• WB

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Western blot - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (AB75829)

All lanes:

Western blot - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (ab75829) at 1/200000 dilution

Lane 1:

Jurkat cell lysate un-treated at 10 µg

Lane 2:

Jurkat cell lysate treated with pervanadate at 10 µg

Secondary

All lanes:

HRP labelled goat anti-rabbit at 1/2000 dilution

Predicted band size: 60 kDa

Observed band size: 76 kDa

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• WB

Lab

Western blot - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (AB75829)

Blocking buffer 5% NFDM/TBST. Diluting buffer 5% NFDM/TBST

All lanes:

Western blot - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (ab75829) at 1/5000 dilution

Lane 1:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates at 15 µg

Lane 2:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates. treated with pervanadate at 50nM for 5 min at 15 µg

Lane 3:

Jurkat (Human T cell leukemia T lymphocyte) whole cell lysates. Treated with pervanadate at 50nM for 5 min. Then the membrane was incubated with phosphatase at 15 µg

Secondary

All lanes:

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

Predicted band size: 60 kDa

Observed band size: 76 kDa

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• Dot

Lab

Dot Blot - Anti-SLP76 (phospho Y145) antibody [EP2853Y] (AB75829)

Dot blot analysis of SLP76 (phospho Y145) phospho peptide (Lane 1) SLP76 Non-phospho peptide (Lane 2) labelling SLP76 (phospho Y145) with ab75829 at a dilution of 1/1000. Peroxidase conjugated goat anti-rabbit IgG (H+L)) was used as the secondary antibody at a dilution of 1/2500.

Blocking and diluting buffer : 5% NFDM/TBST.

Exposure time : 3 minutes.