Anti-FACL4 抗体 [EPR8640]

• WB

Lab

Western blot - Anti-FACL4 antibody [EPR8640] (AB155282)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FACL4 antibody [EPR8640] (ab155282) at 1/10000 dilution

Lane 1:

HeLa whole cell lysate at 20 µg

Lane 2:

HEK293 whole cell lysate at 20 µg

Lane 3:

Human fetal kidney tissue lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugate goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 79 kDa

Observed band size: 79 kDa

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• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FACL4 antibody [EPR8640] (AB155282)

Immunohistochemical analysis of formalin fixed paraffin embedded Human liver carcinoma labelling FACL4 with ab155282 at a concentration of 0.5 µg/ml. The immunostaining was performed on a Ventana DISCOVERY ULTRA (Roche Tissue Diagnostics) instrument with a OptiView DAB IHC Detection Kit. Heat mediated antigen retrieval was performed with DISCOVERY cell conditioning solution (CC1) 100°C, pH8.5 for 32mins.

ab155282 anti FACL4 antibody EPR8640 was incubated for 16mins at 37°C. Sections were counterstained with Hematoxylin II. Image inset shows absence of staining in secondary antibody only control.

Customers are encouraged to optimise antigen retrieval conditions, antibody concentration, incubation times and temperature for best results in their own IHC assay workflow (automated and manual)

• ICC/IF

Lab

Immunocytochemistry/ Immunofluorescence - Anti-FACL4 antibody [EPR8640] (AB155282)

Immunocytochemistry/Immunofluorescence analysis of HeLa cells labelling FACL4 with purified ab155282 at 1/200. Cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100. ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000) was used as the secondary antibody. DAPI (blue) was used as the nuclear counterstain. ab7291, a mouse anti-tubulin (1/1000) and ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000) were also used.

Control 1 : primary antibody (1/200) and secondary antibody, ab150120, an Alexa Fluor^® 594-conjugated goat anti-mouse IgG (1/1000).

Control 2 : ab7291 (1/1000) and secondary antibody, ab150077, an Alexa Fluor^® 488-conjugated goat anti-rabbit IgG (1/1000).

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-FACL4 antibody [EPR8640] (AB155282)

Intracellular Flow Cytometry analysis of 293T cells labelling FACL4 with purified ab155282 at 1/100 (red). Cells were fixed with 2% paraformaldehyde. A FITC-conjugated goat anti-rabbit IgG (1/500) was used as the secondary antibody. Black - Isotype control, rabbit monoclonal IgG. Blue - Unlabelled control, cells without incubation with primary and secondary antibodies.

• Flow Cyt (Intra)

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Flow Cytometry (Intracellular) - Anti-FACL4 antibody [EPR8640] (AB155282)

Intracellular flow cytometric analysis of permeabilized 293T cells labelling FACL4with unpurified ab155282 at a dilution of 1/10 (red) compared to a rabbit IgG (negative) (green).

• IHC-P

Lab

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-FACL4 antibody [EPR8640] (AB155282)

Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) analysis of human hepatocellular carcinoma tissue labelling FACL4 with purified ab155282 at 1/200. Heat mediated antigen retrieval was performed using Tris/EDTA buffer pH 9. ab97051, a HRP-conjugated goat anti-rabbit IgG (H+L) was used as the secondary antibody (1/500). Negative control using PBS instead of primary antibody. Counterstained with hematoxylin.

• IP

Lab

Immunoprecipitation - Anti-FACL4 antibody [EPR8640] (AB155282)

ab155282 (purified) at 1/30 immunoprecipitating FACL4 in HepG2 whole cell lysate.

Lane 1 (input) : HepG2 whole cell lysate (10μg)

Lane 2 (+) : ab155282 + HepG2 whole cell lysate.

Lane 3 (-) : Rabbit monoclonal IgG (ab172730) instead of ab155282 in HepG2 whole cell lysate.

For western blotting, a HRP-conjugated anti-rabbit IgG, specific to the non-reduced form of IgG was used as the secondary antibody (1/1500).

Blocking buffer and concentration : 5% NFDM/TBST.

Diluting buffer and concentration : 5% NFDM /TBST.

All lanes:

Immunoprecipitation - Anti-FACL4 antibody [EPR8640] (ab155282)

Predicted band size: 79 kDa

Observed band size: 79 kDa

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• WB

Lab

Western blot - Anti-FACL4 antibody [EPR8640] (AB155282)

Blocking and dilution buffer : 5% NFDM/TBST.

All lanes:

Western blot - Anti-FACL4 antibody [EPR8640] (ab155282) at 1/50000 dilution

All lanes:

HepG2 whole cell lysate at 20 µg

Secondary

All lanes:

Peroxidase-conjugated goat anti-rabbit IgG (H+L) at 1/1000 dilution

Predicted band size: 79 kDa

Observed band size: 79 kDa

false

• WB

Lab

Western blot - Anti-FACL4 antibody [EPR8640] (AB155282)

Blocking and diluting buffer and concentration : 5% NFDM/TBST.

In Western blot, Anti-GAPDH antibody [EPR16891] - Loading Control (ab181602) staining at 1/1000000 dilution.

Smear bands above the 79 kDa position is acceptable, as FACL4 contains multiple ubiquitination sites.

All lanes:

Western blot - Anti-FACL4 antibody [EPR8640] (ab155282) at 1/1000 dilution

Lane 1:

Human brainstem tissue lysate at 20 µg

Lane 2:

SH-SY5Y (human neuroblastoma epithelial cell) whole cell lysate at 20 µg

Lane 3:

Mouse brain tissue lysate at 20 µg

Lane 4:

Mouse heart tissue lysate at 20 µg

Lane 5:

Mouse kidney tissue lysate at 20 µg

Lane 6:

Rat brain tissue lysate at 20 µg

Lane 7:

Rat heart tissue lysate at 20 µg

Lane 8:

Rat kidney tissue lysate at 20 µg

Secondary

All lanes:

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

Observed band size: 79 kDa

false

Exposure time: 100s

• WB

Unknown

Western blot - Anti-FACL4 antibody [EPR8640] (AB155282)

All lanes:

Western blot - Anti-FACL4 antibody [EPR8640] (ab155282) at 1/10000 dilution

Lane 1:

HepG2 lysate at 10 µg

Lane 2:

HeLa lysate at 10 µg

Lane 3:

293T lysate at 10 µg

Lane 4:

Fetal kidney lysate, human at 10 µg

Predicted band size: 79 kDa

false