MA5-15460

antibody from Invitrogen Antibodies

Targeting: KRT8 CARD2, CK8, CYK8, K2C8, K8, KO

Western blot (Supportive data in Antibodypedia)

ELISA (Recommended by provider)

Immunocytochemistry (Supportive data in Antibodypedia)

Immunohistochemistry (Supportive data in Antibodypedia)

Other assay (Supportive data in Antibodypedia)

Antibody Data

Product number

MA5-15460

Provider

Invitrogen Antibodies

Product name

Cytokeratin 8 Monoclonal Antibody (8A5D12)

Antibody type

Monoclonal

Antigen

Purifed from natural sources

Description

MA5-15460 targets Cytokeratin 8 in indirect ELISA, IF, IHC, and WB applications and shows reactivity with Human samples. The MA5-15460 immunogen is purified recombinant fragment of human Cytokeratin (aa391-483) expressed in E. Coli. MA5-15460 detects Cytokeratin 8 which has a predicted molecular weight of approximately 54kDa.

Reactivity

Human

Host

Mouse

Isotype

IgG

Antibody clone number

8A5D12

Vial size

100 µL

Concentration

Conc. Not Determined

Storage

Store at 4°C short term. For long term storage, store at -20°C, avoiding freeze/thaw cycles.

References

Submitted references

The cultural divide: exponential growth in classical 2D and metabolic equilibrium in 3D environments.

Wrzesinski K, Rogowska-Wrzesinska A, Kanlaya R, Borkowski K, Schwämmle V, Dai J, Joensen KE, Wojdyla K, Carvalho VB, Fey SJ
PloS one 2014;9(9):e106973

Western blot

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Cytokeratin 8 using Cytokeratin 8 monoclonal antibody (Product # MA5-15460) in A549 (1), HeLa (2), MCF-7 (3), A431 (4) and HepG2 (5) cell lysate.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot analysis of Cytokeratin 8 using Cytokeratin 8 monoclonal antibody (Product # MA5-15460) in A549 (1), HeLa (2), MCF-7 (3), A431 (4) and HepG2 (5) cell lysate.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

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 Monoclonal Antibody (8A5D12) (Product # MA5-15460) using 1:2,000 dilution and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177 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)

Main image

Experimental details

Knockdown of KRT8 was achieved by transfecting Caco-2 with KRT8 specific siRNAs (Silencer® select Product # S7969, S7970). Western blot analysis (Fig. a) was performed using Whole cell extracts from the KRT8 knockdown cells (lane 3), non-targeting scrambled siRNA transfected cells (lane 2) and untransfected cells (lane 1). The blot was probed with Cytokeratin 8 Monoclonal Antibody (8A5D12) (Product # MA5-15460, 1:1000) and Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:4000). 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 KRT8.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Western blot was performed using Anti-Cytokeratin 8 Monoclonal Antibody (8A5D12) (Product # MA5-15460) and a 53 kDa band corresponding to KRT8 was observed across the cell lines tested. Whole cell extracts (30 µg lysate) of HeLa (Lane 1), A-431 (Lane 2), MCF7 (Lane 3), HaCaT (Lane 4) and Caco-2 (Lane 5) were electrophoresed using NuPAGE™ 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 the primary antibody (1:1000) and detected by chemiluminescence with Goat anti-Mouse IgG (H+L) Superclonal™ Recombinant Secondary Antibody, HRP (Product # A28177, 1:20,000) using the iBright™ FL1500 Imaging System (Product # A44115). Chemiluminescent detection was performed using SuperSignal™ West Pico PLUS Chemiluminescent Substrate (Product # 34580).

Immunocytochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of methanol-fixed ECA109 cells (left) and HepG2 cells (right) using Cytokeratin 8 monoclonal antibody (Product # MA5-15460) (Green). Blue: DRAQ5 fluorescent DNA dye.

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunofluorescence analysis of KRT8 was performed using 70% confluent log phase HeLa 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 8 Monoclonal Antibody (8A5D12) (Product # MA5-15460) at 1:100 in 0.1% BSA, incubated at 4 degree celsius overnight and then labeled with Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor Plus 488 (Product # A32723, 1:2000), 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). Panel d represents the merged image showing Intermediate filaments localization. Panel e represents control cells with no primary antibody to assess background. The images were captured at 60X magnification.

Immunohistochemistry

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of paraffin-embedded human breast carcinoma (A), lung cancer (B) and ovarian cancer tissue (C), showing membrane and cytoplasmic localization using Cytokeratin 8 monoclonal antibody (Product # MA5-15460) followed with DAB staining

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Immunohistochemical analysis of paraffin-embedded human kidney tissues using Cytokeratin 8 monoclonal antibody (Product # MA5-15460).

Other assay

Supportive validation

Submitted by

Invitrogen Antibodies (provider)

Main image

Experimental details

Figure 4 Immunohistochemical staining of tubulin and keratin. C3A cells were grown using either classical 2D flat culture techniques or as 3D microgravity spheroid techniques. Cells from 2D cultures were fixed directly while cells grown in 3D were fixed and sectioned. a, c, e and g: HepG2/C3A exponentially growing cells (2D), b, d, f and h HepG2/C3A cells at dynamic equilibrium. a and b: acetylated tubulin to highlight filaments, c and d: staining of alpha-tubulin; e and f keratin 8. All photographs were made at the same magnification: the bar in h indicates 25 uM.