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Western blotAntibody data
- Antibody Data
- Antigen structure
- References [0]
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- Validations
- Western blot [1]
- Immunocytochemistry [1]
- Immunohistochemistry [1]
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- Product number
- PA5-32464 - Provider product page
- Provider
- Invitrogen Antibodies
- Product name
- Cytokeratin 20 Polyclonal Antibody
- Antibody type
- Polyclonal
- 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 adenocarcinoma.
- Reactivity
- Human
- Host
- Rabbit
- Isotype
- IgG
- Vial size
- 500 µL
- Storage
- -20° C, Avoid Freeze/Thaw Cycles
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Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Western Blot was performed using Cytokeratin 20 Polyclonal Antibody (Product # PA5-32464) and a 48 kDa band corresponding to Cytokeratin 20 was observed across all the cell lines tested. Whole cell extracts (30 µg lysate) of HT-29 (Lane 1), HCT116 (Lane 2), Caco-2 (Lane 3), MCF-7 (Lane 4), SH-SY5Y (Lane 5) were electrophoresed using NuPAGE™ 12% Bis-Tris Protein Gel (Product # NP0342BOX). 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 (1:1000 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).
Supportive validation
- Submitted by
- Invitrogen Antibodies (provider)
- Main image
- Experimental details
- Immunofluorescence analysis of Cytokeratin 20 was performed using HT-29 and SH-SY5Y 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 1 hour at room temperature. The cells were labeled with Cytokeratin 20 Polyclonal Antibody (Product # PA5-32464) at 1:200 dilution in 0.1% BSA and incubated overnight at 4 degree and then labeled with Goat anti-Rabbit IgG (H+L) Superclonal™ Secondary Antibody, Alexa Fluor® 488 conjµgate (Product # A27034), (1:2000 dilution) for 45 minutes at room temperature (Panel a: green) in HeLa cells. 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). Panel d represents the merged image of HT-29 cells showing cytoskeletal and cytosolic localization for Cytokeratin 20. Panel e represents SH-SY5Y cells with no expression. 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 Keratin 20 using anti-Keratin 20 Polyclonal Antibody (Product # PA5-32464) in Colon Adenocarcinoma Cancer Tissue. The recommened dilution for this antibody in immunohistochemistry applications is 1:200.
