- PA5-34762 - Invitrogen Antibodies KRT8 antibody

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Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot analysis of Cytokeratin 8 using 30 µg of A) HeLa and B) HepG2 lysate. Samples were loaded onto a 10% SDS-PAGE gel and probed with a Cytokeratin 8 polyclonal antibody (Product # PA5-34762) at a dilution of 1:10,000.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: CRISPR-Cas9 mediated genome editing ofCytokeratin 8 (as confirmed by next generation sequencing) was achieved by using LentiArray™ Lentiviral sgRNA (Product # A32042, AssayID CRISPR745510_LV) and LentiArray Cas9 Lentivirus (Product # A32064). Fig (a) Western blot analysis of Cytokeratin 8 was performed by loading 30 µg of HeLa Wild Type (Lane 1), HeLa Cas9 (Lane 2) and HeLa Cas9 cells transduced with Cytokeratin 8 Lentiviral sgRNA (Lane 3) whole cell extracts. The samples were electrophoresed using NuPAGE™ Novex™ 4-12% Bis-Tris Protein Gel (Product # NP0322BOX). Resolved proteins were then transferred onto a nitrocellulose membrane (Product # IB23001) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with Anti-Cytokeratin 8 Polyclonal Antibody (Product # PA5-34762) using 1:10,000 dilution and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036 1:5,000 dilution).Chemiluminescent detection was performed using Novex® ECL Chemiluminescent Substrate Reagent Kit (Product # WP20005). A loss of signal in sgRNA transduced cells using the LentiArray™ CRISPR product line confirms that antibody is specific toCytokeratin 8 (Fig (b)).

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using Cytokeratin 8 Polyclonal Antibody (Product # PA5-34762). Sample (30 µg of whole cell lysate). Lane A: JC. 10% SDS PAGE. Cytokeratin 8 Polyclonal Antibody (Product # PA5-34762) diluted at 1:10,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western Blot using Cytokeratin 8 Polyclonal Antibody (Product # PA5-34762). Various whole cell extracts (30 µg) were separated by 10% SDS-PAGE, and the membrane was blotted with Cytokeratin 8 Polyclonal Antibody (Product # PA5-34762) diluted at 1:10,000. The HRP-conjugated anti-rabbit IgG antibody was used to detect the primary antibody.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Knockdown of Cytokeratin 8 was achieved by transfecting Caco-2 with Cytokeratin 8 specific siRNAs (Silencer® select Product # S7969, S7970). Western blot analysis (Fig. a) was performed using Whole cell extracts from the Cytokeratin 8 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Cytokeratin 8 Polyclonal Antibody (Product # PA5-34762,1:10000 dilution) and Goat anti-Rabbit IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A27036, 1:4000 dilution). Densitometric analysis of this western blot is shown in histogram (Fig. b). Decrease in signal upon siRNA mediated knock down confirms that antibody is specific to Cytokeratin 8.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Western blot was performed using Anti-Cytokeratin 8 Polyclonal Antibody(Product # PA5-34762) and 53, 57kDa bands corresponding to Cytokeratin 8 was observed across the cell lines tested except IMR-32, SH-SY5Y and SK-MEL-31. Whole cell extracts (30 µg lysate) of Caco-2 (Lane 1), HaCaT (Lane 2), MCF-7 (Lane 3), IMR-32 (Lane 4), SH-SY5Y (Lane 5), and SK-MEL-31 (Lane 6) were electrophoresed using NuPAGE™ 4-12% Bis-Tris Protein Gel (Product # NP0321BOX). Resolved proteins were then transferred onto a Nitrocellulose membrane (Product # LC2002) by iBlot® 2 Dry Blotting System (Product # IB21001). The blot was probed with the primary antibody (1:10000 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)
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Experimental details: Immunocytochemistry-Immunofluorescence analysis of Cytokeratin 8 was performed in HeLa cells fixed in 4% paraformaldehyde at RT for 15 min. Green: Cytokeratin 8 Polyclonal Antibody (Product # PA5 34762) diluted at 1:500. Blue: Hoechst 33342 staining.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunofluorescence analysis of Cytokeratin 8 was performed using 70% confluent log phase Caco-2 cells. The cells were fixed with ice-cold acetone at 4°C for 5 minutes and blocked with 2% BSA for 45 minutes at room temperature. The cells were labeled with Cytokeratin 8 Polyclonal Antibody (Product # PA5-34762) at 1:200 dilution in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, Alexa Fluor® 488 conjugate (Product # A28175), (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 cytoskeletal localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Supportive validation

Submitted by: Invitrogen Antibodies (provider)
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Experimental details: Immunohistochemical analysis of paraffin-embedded Gastric ca, using Cytokeratin 8 (Product # PA5-34762) antibody at 1:500 dilution. Antigen Retrieval: EDTA based buffer, pH 8.0, 15 min.

PA5-34762