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

ab130751 staining Villin in mouse intestine tissue sections by Immunohistochemistry (IHC-P - paraformaldehyde-fixed, paraffin-embedded sections). Tissue was fixed with paraformaldehyde and blocked with 5% serum for 30 minutes at 25°C; antigen retrieval was by heat mediation in 10mM citrate buffer, pH 6. Samples were incubated with primary antibody (1/1000) for 16 hours at 4°C. An Alexa Fluor^® 568-conjugated goat anti-rabbit IgG monoclonal (1/250) was used as the secondary antibody.

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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

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 >5,000 events were collected using a 20mW Argon ion laser (488nm) and 525/30 bandpass filter.

Anti-Villin antibody [SP145] (ab130751) at 1/100 dilution + HT-29 cell lysate

Predicted band size: 93 kDa