Anti-Syk antibody [EP573Y]

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [EP573Y] (AB40781)

ab40781 was shown to react with SYK in wild-type THP-1 cells in immunocytochemistry with loss of signal observed in SYK knockout cell line ab288700. Wild-type and knockout cells were mixed and pelleted at a 1 : 1 ratio on coverslips. The cells were fixed with 4% paraformaldehyde (15 min) then permeabilized with 0.1% Triton X-100 (10min) and then blocked with 1x PBS, 0.01% Triton X-100, 5% BSA, 5% NGS. The cells were then incubated with ab40781 at dilution overnight at 4°C followed by a further incubation at room temperature for 1h with a goat anti-rabbit secondary antibody to (Alexa Fluor® 555) at 0.5 μg/ml. Acquisition of the green (wild-type), red (antibody staining) and far-red (knockout) channels was performed. Representative grayscale images of the red channel are shown. Wild-type and knockout cells are outlined with yellow and magenta dashed line, respectively. Schematic representation of the mosaic strategy used is shown on the bottom-right panel. Image was acquired with a Zeiss(LSM-880).

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

• IHC-P

Unknown

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Syk antibody [EP573Y] (AB40781)

Immunohistochemistry of paraffin embedded Human spleen tissue section labelling Syk with ab40781 at 1 : 5000 dilution (0.44 μg/ml). Heat mediated antigen retrieval was performed using ab93684 (Tris/EDTA buffer, pH 9.0). A ready to use ImmunoHistoProbe one step HRP Polymer (ready to use) was used as a secondary antibody at 1 : 0 dilution. PBS instead of primary antibody was used for negative control. Hematoxylin was used as a counterstain.

• WB

Lab

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

False colour image of Western blot : Anti-Syk antibody [EP573Y] staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40781 was shown to bind specifically to Syk. A band was observed at 70/72 kDa in wild-type HEK-293T cell lysates with no signal observed at this size in SYK knockout cell line ab282649 (knockout cell lysate ab283048). To generate this image, wild-type and SYK knockout HEK-293T 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 (IRDye^® 800CW) preabsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preabsorbed (ab216776) at 1/20000 dilution.

All lanes:

Western blot - Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution

Lane 1:

Wild-type HEK-293T cell lysate at 20 µg

Lane 2:

SYK knockout HEK-293T cell lysate at 20 µg

Lane 2:

Western blot - Human SYK knockout HEK-293T cell line (<a href='/en-us/products/cell-lines/human-syk-knockout-hek-293t-cell-line-ab282649'>ab282649</a>)

Predicted band size: 72 kDa

Observed band size: 70 kDa

false

• WB

Unknown

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

Blocking/Diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution

All lanes:

Human bone marrow lysates at 15 µg

Secondary

All lanes:

Goat Anti-Rabbit IgG (HRP) with minimal cross-reactivity with human IgG at 1/2000 dilution

Predicted band size: 72 kDa

Observed band size: 72 kDa

false

• WB

Lab

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

Lane 1 : Wild-type HAP1 cell lysate (40 μg)
Lane 2 : SYK knockout HAP1 cell lysate (40 μg)
Lane 3 : K562 cell lysate (40 μg)
Lane 4 : Raji cell lysate (40 μg)
Lanes 1 - 4 : Merged signal (red and green). Green - ab40781 observed at 75 kDa. Red - loading control, ab8245, observed at 37 kDa.

ab40781 was shown to specifically react with Syk when Syk knockout samples were used. Wild-type and Syk knockout samples were subjected to SDS-PAGE. ab40781 and ab8245 (loading control to GAPDH) were diluted at 1/5000 and 1/10000 dilution respectively and incubated overnight at 4C. Blots were developed with Goat anti-Rabbit IgG H&L (IRDye^® 800CW) preadsorbed (ab216773) and Goat anti-Mouse IgG H&L (IRDye^® 680RD) preadsorbed (ab216776) secondary antibodies at 1/10000 dilution for 1 hour at room temperature before imaging.

All lanes:

Western blot - Anti-Syk antibody [EP573Y] (ab40781)

Predicted band size: 72 kDa

false

• WB

Lab

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

Western blot : Anti-SYK antibody [EP573Y] (ab40781) staining at 1/1000 dilution, shown in green; Mouse anti-GAPDH antibody [6C5] (ab8245) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab40781 was shown to bind specifically to SYK. A band was observed at 72 kDa in wild-type THP-1 cell lysates with no signal observed at this size in SYK knockout cell line ab288700 (knockout cell lysate ab289593). To generate this image, wild-type and SYK knockout THP-1 cell lysates were analysed. First, samples were run on an SDS-PAGE gel then transferred onto a nitrocellulose membrane. Membranes were blocked in 5 % 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-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution

Lanes 1 - 4:

Western blot at 20 µg

Lane 2:

Western blot - Human SYK knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-syk-knockout-thp-1-cell-line-ab288700'>ab288700</a>)

Secondary

All lanes:

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

Predicted band size: 72 kDa

Observed band size: 72 kDa

false

• WB

Lab

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

ab40781 was shown to react with SYK in wild-type THP-1 cells in Western blot with loss of signal observed in SYK knockout cell line ab288700. Wild-type THP-1 and SYK knockout cell lysates were subjected to SDS-PAGE. Membranes were blocked in 5% milk in TBST for 1 hr before incubation with ab40781 overnight at 4 °C at a 1/1000 dilution. Blots were incubated with secondary antibodies at 0.2ug/mL before imaging.

This data was provided by YCharOS Inc., an open science company with the mission of characterizing commercially available antibody reagents for all human proteins. Abcam and YCharOS are working together to help address the reproducibility crisis by enabling the life science community to better evaluate commercially available antibodies.

All lanes:

Western blot - Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution

Lane 1:

Wild-type THP-1 lysate at 20 µg

Lane 2:

Western blot - Human SYK knockout THP-1 cell line (<a href='/en-us/products/cell-lines/human-syk-knockout-thp-1-cell-line-ab288700'>ab288700</a>) at 20 µg

Observed band size: 72 kDa

false

• WB

Unknown

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

Blocking/Diluting buffer : 5% NFDM/TBST

All lanes:

Western blot - Anti-Syk antibody [EP573Y] (ab40781) at 1/1000 dilution

Lane 1:

Raji (Human Burkitt's lymphoma B lymphocyte) whole cell lysates at 15 µg

Lane 2:

Daudi (Human Burkitt's lymphoma lymphoblast) whole cell lysates at 15 µg

Lane 3:

WEHI-231 (Mouse B cell lymphoma B lymphocyte ) whole cell lysates 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 1/20000 dilution

Predicted band size: 72 kDa

Observed band size: 72 kDa

false

• WB

Lab

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Syk antibody [EP573Y] (ab40781) at 1/2000 dilution

All lanes:

Rat bone marrow 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: 72 kDa

false

Exposure time: 50s

• WB

Lab

Western blot - Anti-Syk antibody [EP573Y] (AB40781)

Blocking and diluting buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-Syk antibody [EP573Y] (ab40781) at 1/5000 dilution

All lanes:

Mouse bone marrow 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: 72 kDa

false

Exposure time: 3min