Anti-Villin antibody [SP145] - BSA and Azide free

5 product images

• 

  Immunoprecipitation - Anti-Villin antibody [SP145] - BSA and Azide free (ab245749)

  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 Anti-Villin antibody [SP145] ab130751.

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

  Band: 93kDa; Villin

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (Anti-Villin antibody [SP145] ab130751).

  All lanes: Immunoprecipitation - Anti-Villin antibody [SP145] (Anti-Villin antibody [SP145] ab130751)

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 93 kDa

  Exposure time: 16min

• 

  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Villin antibody [SP145] - BSA and Azide free (ab245749)

  Overlay histogram showing Caco 2 cells stained with Anti-Villin antibody [SP145] 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 (Anti-Villin antibody [SP145] ab130751, 1/100 dilution) for 30 min at 22�C. The secondary antibody used was Alexa Fluor� 488 goat anti-rabbit IgG (H&L) (Goat Anti-Rabbit IgG H&L (Alexa Fluor® 488) ab150077) at 1/2000 dilution for 30 min at 22�C. Isotype control antibody (black line) was rabbit IgG (monoclonal) (1?g/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.This data was developed using the same antibody clone in a different buffer formulation containing PBS#44; BSA#44; and sodium azide (Anti-Villin antibody [SP145] ab130751)

• 

  Flow Cytometry (Intracellular) - Anti-Villin antibody [SP145] - BSA and Azide free (ab245749)

  Anti-Villin antibody [SP145] 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.

  This data was developed using the same antibody clone in a different buffer formulation containing PBS#44; BSA#44; and sodium azide (Anti-Villin antibody [SP145] ab130751)

• 

  Multiplex immunohistochemistry - Anti-Villin antibody [SP145] - BSA and Azide free (ab245749)

  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 (Anti-liver FABP antibody [EPR20464] - BSA and Azide free ab240401, green; Opal™520) and anti-MUC2 (Anti-MUC2 antibody [EPR23479-47] 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 (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), Anti-liver FABP antibody [EPR20464] - BSA and Azide free ab240401 (1/8000 dilution), and Anti-MUC2 antibody [EPR23479-47] 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.
  
  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA and sodium azide (Anti-Villin antibody [SP145] ab130751).

• 

  Flow Cytometry (Intracellular) - Anti-Villin antibody [SP145] - BSA and Azide free (ab245749)

  This data was developed using the same antibody clone in a different buffer formulation containing PBS, BSA, glycerol, and sodium azide (Anti-Villin antibody [SP145] ab130751).
  Flow cytometry overlay histogram showing left HT-29 positive cells and right negative PANC-1 stained with Anti-Villin antibody [SP145] 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 (Anti-Villin antibody [SP145] 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.