Anti-Syk antibody [SYK-01]

• Flow Cyt

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Flow Cytometry - Anti-Syk antibody [SYK-01] (AB3993)

Overlay histogram showing Ramos (Human Burkitt's lymphoma cell line) cells stained with ab3993 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.1% PBS-Tween for 20 min. The cells were then incubated in 1x PBS / 10% normal goat serum / 0.3M glycine to block non-specific protein-protein interactions followed by the antibody (ab3993, 1μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight^® 488 goat anti-mouse IgG (H+L) (ab96879) at 1/500 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in Ramos cells fixed with 80% methanol (5 min)/permeabilized with 0.1% PBS-Tween for 20 min used under the same conditions.

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-Syk antibody [SYK-01] (AB3993)

ab3993 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 ab3993 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.

• WB

Lab

Western blot - Anti-Syk antibody [SYK-01] (AB3993)

ab3993 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 ab3993 overnight at 4 °C at a 1/500 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 [SYK-01] (ab3993) at 1/500 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

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• WB

Lab

Western blot - Anti-Syk antibody [SYK-01] (AB3993)

False colour image of Western blot : Anti-Syk antibody [SYK-01] staining at 1 ug/ml, shown in green; Rabbit Anti-GAPDH antibody [EPR16891] (ab181602) loading control staining at 1/20000 dilution, shown in red. In Western blot, ab3993 was shown to bind specifically to Syk. A band was observed at 72/73 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-Mouse IgG H&L (IRDye^® 800CW) preabsorbed (ab216772) and Goat anti-Rabbit IgG H&L (IRDye^® 680RD) preabsorbed (ab216777) at 1/20000 dilution.

All lanes:

Western blot - Anti-Syk antibody [SYK-01] (ab3993) at 1 µg/mL

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>)

Lane 3:

HAP1 cell lysate at 20 µg

Predicted band size: 72 kDa

Observed band size: 72 kDa,73 kDa

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• WB

Supplier Data

Western blot - Anti-Syk antibody [SYK-01] (AB3993)

Lanes 1 - 2:

Western blot - Anti-Syk antibody [SYK-01] (ab3993) at 2 µg/mL

Lanes 3 - 4:

anti-Human Cytokeratin 18

Lane 5:

Western blot - Anti-Syk antibody [SYK-01] (ab3993)

Lane 1:

Ramos (human Burkitt's lymphoma cell line) cell lysate

Lane 2:

RBL (rat basophilic leukemia cell line) cell lysate

Lanes 3 - 4:

HeLa (human epithelial cell line from cervix adenocarcinoma) cell lysate

Lane 5:

negative control

Predicted band size: 72 kDa

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• WB

CiteAb

Western blot - Anti-Syk antibody [SYK-01] (AB3993)

Syk western blot using anti-Syk antibody [SYK-01] ab3993. Publication image and figure legend from Ho, C. M., Donovan-Banfield, I. Z., et al., 2016, PLoS One, PubMed 26930276.

ab3993 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab3993 please see the product overview.

Investigation of roles for SYK and GCK in HCMV infected cells.(A and B) Analysis of siRNA treated cells infected with AD169. HFF were treated with the siRNAs indicated in each figure for 72 hours and infected with 1x105 plaque forming units (p.f.u.) of HCMV. At 96 h.p.i cell lysates were prepared for western blot (Fig 2A) and viral supernatants were harvested for virus titration (Fig 2B). In Fig 2B viral titre is expressed as plaque forming units/ml (p.f.u./ml) and the mean and standard deviation of 3 experiments is shown. (C) Analysis of HCMV infected cells treated with inhibitors of GCK. HFF cells were infected with AD169 at an MOI of 1 then treated with 1μM of the drug indicated in the figure or the equivalent volume of DMSO. Viral supernatants were harvested at the indicated time points and viral titre (p.f.u./ml) at each time point was determined. (D and E) Western blot of lysate from uninfected or infected cell lines. Unless stated otherwise, all lysates are from HFF cells. Where indicated, HFF cells were either uninfected or infected with AD169 at an MOI of 1 then treated with either 1μM BAY61-3606, 1μM Maribavir or the equivalent volume of DMSO. Cell lysates were prepared for western blot at 72 hours post infection. In each panel showing western blot proteins recognized by the antibodies used in each experiment are indicated to the right of each figure and the positions of molecular weight markers (kDa) are indicated to the left of each figure.

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