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Western blot
ImmunoprecipitationAntibody data
- Antibody Data
- Antigen structure
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- Validations
- Western blot [2]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
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- Product number
- MA5-16408 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Villin Monoclonal Antibody (SP145)
- Antibody type
- Monoclonal
- Antigen
- Synthetic peptide
- Description
- Heat-mediated antigen retrieval is recommended prior to staining, using a 10mM citrate buffer, pH 6.0, for 10 minutes followed by cooling at room temperature for 20 min. Following antigen retrieval, incubate samples with primary antibody for 10 min at room temperature. A suggested positive control is colon, small intestine or kidney tissue.
- Reactivity
- Human, Mouse
- Host
- Rabbit
- Isotype
- IgG
- Antibody clone number
- SP145
- Vial size
- 500 µL
- Concentration
- 0.108 mg/mL
- Storage
- 4° C, do not freeze
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot was performed using Anti-Villin Monoclonal Antibody (SP145) (Product # MA5-16408) and a 92 kDa band corresponding to Villin was observed across cell lines and tissues tested. Whole cell extracts (30 µg µg lysate) of Caco-2 (Lane 1), COLO 205 (Lane 2), HT-29 (Lane 3), Jurkat (Lane 4) and PANC-1 (Lane 5) as seen in Fig (a) .Tissue Extracts of Mouse Liver (Lane 1), Mouse Kidney (Lane 2), Mouse Colon (Lane 3) and Mouse Jejunum (Lane 4) as seen in fig (b) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:500 dilution) and detected by chemiluminescence with Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution) using the iBright FL 1000 (Product # A32752). Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005).(Ref-http://dx.doi.org/10.1016/j.cbpa.2016.08.016, https://doi.org/10.1371/journal.pone.0084221).
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western blot analysis of HT-29 Cells using anti-Villin Monoclonal Antibody (Product # MA5-16408). The recommened dilution for this antibody in western blot applications is 1:100.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Villin was performed using 70 confluent log phase Caco-2 cells. The cells were fixed with 4% paraformaldehyde for 10 minutes, permeabilized with 0.1% Triton™ X-100 for 15 minutes, and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Villin Monoclonal Antibody (SP145) (Product # MA5-16408) at 1:100 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Donkey anti-Rabbit IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32790), (1:2000 dilution), for 45 minutes at room temperature (Panel a: Green). Nuclei (Panel b: Blue) were stained with ProLong™ Diamond Antifade Mountant with DAPI (Product # P36962). F-actin (Panel c: Red) was stained with Rhodamine Phalloidin (Product # R415, 1:300 dilution). Panel d represents the merged image showing actin filaments localization. Panel e represents no expression in PANC-1. Panel f represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunohistochemical analysis of Villin using anti-Villin Monoclonal Antibody (Product # MA5-16408) in Colon Cancer Tissue. The recommened dilution for this antibody in immunohistochemistry applications is 1:100.
