Anti-CYLD antibody [733] - N-terminal

• ICC/IF

Supplier Data

Immunocytochemistry/ Immunofluorescence - Anti-CYLD antibody [733] - N-terminal (AB200476)

Immunofluorescence analysis of 70% confluent log phase SHSY5Y cells fixed with 4% paraformaldehyde for 15 minutes, permeabilized with 0.25% Triton™ X-100 for 10 minutes, and blocked with 5% BSA for 1 hour at room temperature. Labeling CYLD with ab200476 CYLD at 1 μg/mL in 1% BSA and incubated for 3 hours at room temperature and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjugate at a dilution of 1/2000 for 45 minutes at room temperature (Panel a : green). Nuclei (Panel b : blue) were stained with SlowFade® Gold Antifade Mountant with DAPI. F-actin (Panel c : red) was stained with Alexa Fluor® 555 Rhodamine Phalloidin at 1/300 dilution. Panel d is a merged image showing cytoplasmic localization. Panel e is a no primary antibody control. The images were captured at 60X magnification.

• IHC-P

Supplier Data

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-CYLD antibody [733] - N-terminal (AB200476)

Immunohistochemical analysis of of paraffin-embedded mouse brain tissue (right) compared to a negative control without primary antibody (left) labeling CYLD with ab200476 at 1/20 dilution.

To expose target proteins, antigen retrieval was performed using 10 mM sodium citrate (pH 6.0), microwaved for 8-15 min. Following antigen retrieval, tissues were blocked in 3% H2O2-methanol for 15 min at room temperature, washed with ddH2O and PBS, and then probed with ab200476 diluted in 3% BSA-PBS at a dilution of 1/20 overnight at 4°C in a humidified chamber. Tissues were washed extensively in PBST and detection was performed using an HRP-conjugated secondary antibody followed by colorimetric detection using a DAB kit. Tissues were counterstained with hematoxylin and dehydrated with ethanol and xylene to prep for mounting.

• WB

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Western blot - Anti-CYLD antibody [733] - N-terminal (AB200476)

Knockdown of CYLD was observed by transfecting U-87 MG with CYLD specific siRNAs. Western blot analysis (Fig. a) was performed using Whole cell extracts from the CYLD knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to CYLD.

All lanes:

Western blot - Anti-CYLD antibody [733] - N-terminal (ab200476) at 1/1000 dilution

Lane 1:

U-87 MG transfected with CYLD specific siRNAs

Lane 2:

non-targeting scrambled siRNA transfected cells

Lane 3:

untransfected cells

Secondary

All lanes:

Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP at 1/4000 dilution

Predicted band size: 107 kDa

Observed band size: ~107 kDa

false

• WB

Supplier Data

Western blot - Anti-CYLD antibody [733] - N-terminal (AB200476)

All lanes:

Western blot - Anti-CYLD antibody [733] - N-terminal (ab200476) at 2 µg/mL

All lanes:

Jurkat cell lysate

Predicted band size: 107 kDa

false