Anti-Villin antibody [SP145]

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Villin antibody [SP145] (AB130751)

Overlay histogram showing Caco 2 cells stained with ab130751 (red line). The cells were fixed with 80% methanol (5 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 (ab130751 1/100 dilution) for 30 min at 22°C. The secondary antibody used was Alexa Fluor^® 488 goat anti-rabbit IgG (H&L) (ab150077) at 1/2000 dilution for 30 min at 22°C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1μg/1x10⁶ cells) used under the same conditions. Unlabelled sample (blue line) was also used as a control. Acquisition of >5000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

• IHC-P

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Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Villin antibody [SP145] (AB130751)

ab130751 at 1/100 dilution staining Villin in formalin-fixed paraffin-embedded Human colon tissue by Immunohistochemistry.

• mIHC

Lab

Multiplex immunohistochemistry - Anti-Villin antibody [SP145] (AB130751)

Fluorescence multiplex immunohistochemical analysis of the human colon (Formalin/PFA-fixed paraffin-embedded sections). Panel A : merged staining of anti-Villin (ab245749, gray; Opal™690), anti-liver FABP (ab240401, green; Opal™520) and anti-MUC2 (ab272692, red; Opal™570) on human colon. Panel B : anti-liver FABP stained on enterocytes. Panel C : anti-Villin stained on apical border. Panel D : anti-MUC2 stained on goblet cells. Rabbit specific IHC polymer detection kit HRP/DAB (ab209101) was used as a secondary antibody. The immunostaining was performed on a Leica Biosystems BOND® RX instrument with an Opal™ 4-color kit. The section was incubated in three rounds of staining : in the order of ab245749 (1/1000 dilution), ab240401 (1/8000 dilution), and ab272692 (1/5000 dilution) for 30 mins at room temperature. Each round was followed by a separate fluorescent tyramide signal amplification system. Heat mediated antigen retrieval with Tris-EDTA buffer (pH 9.0, epitope retrieval solution2) for 20 mins. DAPI (blue) was used as a nuclear counter stain. Image acquisition was performed with Leica SP8 confocal microscope.

• Flow Cyt (Intra)

Lab

Flow Cytometry (Intracellular) - Anti-Villin antibody [SP145] (AB130751)

Flow cytometry overlay histogram showing left HT-29 positive cells and right negative PANC-1 stained with ab130751 (red line). The cells were fixed with 80% methanol (5 min) and then permeabilised with 0.1% PBS-Triton X-100 for 15 min. The cells were then incubated in 1x PBS containing 10% normal goat serum to block non-specific protein-protein interaction followed by the antibody (ab130751) (1x 106 in 100μl at 0.2μg/ml (1/11200)) for 30min at 22°C.

The secondary antibody Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) preadsorbed was incubated at 1/4000 for 30min at 22°C

Isotype control antibody (black line) was Recombinant Rabbit IgG, monoclonal [EPR25A] - Isotype Control used at the same concentration and conditions as the primary antibody. Unlabelled sample (blue line) was also used as a control.

Acquisition of >5000 events were collected using a 50 mW Blue laser (488nm) and 525/40 bandpass filter.

This antibody gave a positive signal in HT-29 Fixed with 4% formaldehyde (10 min) / permeabilised with 0.1% PBS-Triton X-100 for 15 min under the same conditions.

• IP

Lab

Immunoprecipitation - Anti-Villin antibody [SP145] (AB130751)

Villin was immunoprecipitated using 0.5mg SW480 whole cell extract, 5μg of Rabbit polyclonal to Villin and 50μl of protein G magnetic beads (+). No antibody was added to the control (-).

The antibody was incubated under agitation with Protein G beads for 10min, SW480 whole cell extract lysate diluted in RIPA buffer was added to each sample and incubated for a further 10min under agitation.

Proteins were eluted by addition of 40μl SDS loading buffer and incubated for 10min at 70°C; 10μl of each sample was separated on a SDS PAGE gel, transferred to a nitrocellulose membrane, blocked with 5% BSA and probed with ab130751.

Secondary : Mouse monoclonal [SB62a] Secondary Antibody to Rabbit IgG light chain (HRP) (ab99697).

Band : 93kDa; Villin

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Immunoprecipitation - Anti-Villin antibody [SP145] (ab130751)

Predicted band size: 93 kDa

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Exposure time: 16min

• WB

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Western blot - Anti-Villin antibody [SP145] (AB130751)

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Western blot - Anti-Villin antibody [SP145] (ab130751) at 1/100 dilution

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HT-29 cell lysate

Predicted band size: 93 kDa

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