Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker

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  This image is a courtesy of Anonymous customer review

  Western blot - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  All lanes: Western blot - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023) at 1/2000 dilution

  Lane 1: Lysate prepared from human prostate cancer LNCaP cells at 50 µg

  Lane 2: Lysate prepared from human prostate cancer PC3 cells at 50 µg

  Secondary

  All lanes: HRP-conjugated goat polyclonal to mouse IgG at 1/10000 dilution

  Developed using the ECL technique.

  Performed under reducing conditions.

  Predicted band size: 53 kDa

  Observed band size: 26 kDa, 50 kDa, 65 kDa

  Exposure time: 4min

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  Western blot - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  ** Lanes 1 - 4:** Merged signal (red and green). Green - ab9023 observed at 55 kDa. Red - loading control, Anti-GAPDH antibody [EPR16891] - Loading Control ab181602 observed at 37 kDa.
  ab9023 was shown to react with Cytokeratin 8 in wild-type HeLa cells. Loss of signal was observed when knockout cell line Human KRT8 (Cytokeratin 8) knockout HeLa cell line ab255400 (knockout cell lysate Human KRT8 (Cytokeratin 8) knockout HeLa cell lysate ab263785) was used. Wild-type and Cytokeratin 8 knockout samples were subjected to SDS-PAGE. ab9023 and Anti-GAPDH antibody EPR16891] - Loading Control (Anti-GAPDH antibody [EPR16891] - Loading Control ab181602) were incubated overnight at 4°C at 1 in 1000 dilution and 1 in 20000 dilution respectively. Blots were developed with Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) and Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed (Goat Anti-Rabbit IgG H&L (IRDye® 680RD) preadsorbed ab216777) secondary antibodies at 1 in 20000 dilution for 1 hour at room temperature before imaging.

  All lanes: Western blot - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023) at 1/1000 dilution

  Lane 1: A431 cell lysate at 20 µg

  Lane 2: MCF7 cell lysate at 20 µg

  Lane 2: Western blot - Human KRT8 (Cytokeratin 8) knockout HeLa cell line (Human KRT8 (Cytokeratin 8) knockout HeLa cell line ab255400)

  Lane 3: Wild-type HeLa cell lysate at 20 µg

  Lane 4: KRT8 knockout HeLa cell lysate at 20 µg

  Secondary

  All lanes: Western blot - Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed (Goat anti-Mouse IgG H&L (IRDye® 800CW) preadsorbed ab216772) at 1/20000 dilution

  Predicted band size: 53 kDa

  Observed band size: 37 kDa

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  Flow Cytometry - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  Overlay histogram showing MCF-7 cells stained with ab9023 (red line). The cells were fixed with 4% paraformaldehyde (10 min) and then permeabilized with 0.5% 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 (ab9023, 2μg/1x10⁶ cells) for 30 min at 22°C. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (Goat Anti-Mouse IgG H&L (DyLight® 488) preadsorbed ab96879) at 1/250 dilution for 30 min at 22°C. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (Mouse IgG1, Kappa Monoclonal [B11/6] - Isotype Control ab91353, 2μg/1x10⁶ cells) used under the same conditions. Acquisition of >5,000 events was performed. This antibody gave a positive signal in MCF-7 cells fixed with methanol (5 min)/permeabilized in 0.5% PBS-Tween used under the same conditions.

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  Western blot - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  The image shows different concentration of total protein from normal and tumour colon tissues.

  This picture was kindly supplied as part of the review submitted by Semona Rupchand.

  The image shows different concentration of total protein from normal and tumour colon tissues.

  This picture was kindly supplied as part of the review submitted by Semona Rupchand.

  All lanes: Western blot - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  Predicted band size: 53 kDa

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  Immunohistochemistry (Frozen sections) - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  Immunohistochemistry analysis of frozen section of human colon labeling Cytokeratin 8 with ab9023.

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  Immunohistochemistry on paraffin section of human colon

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  Immunohistochemistry (Formalin/PFA-fixed paraffin-embedded sections) - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  ab9023 staining human normal liver parenchima tissue. Staining is localized to cell membrane.
  Left panel: with primary antibody at 4 ug/ml. Right panel: isotype control.
  Sections were stained using an automated system DAKO Autostainer Plus , at room temperature. Sections were rehydrated and antigen retrieved with the Dako 3-in-1 AR buffer EDTA pH 9.0 in a DAKO PT Link. Slides were peroxidase blocked in 3% H2O2 in methanol for 10 minutes. They were then blocked with Dako Protein block for 10 minutes (containing casein 0.25% in PBS) then incubated with primary antibody for 20 minutes and detected with Dako Envision Flex amplification kit for 30 minutes. Colorimetric detection was completed with Diaminobenzidine for 5 minutes. Slides were counterstained with Haematoxylin and coverslipped under DePeX. Please note that for manual staining we recommend to optimize the primary antibody concentration and incubation time (overnight incubation), and amplification may be required.

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  Immunohistochemistry (Frozen sections) - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  Immunohistochemistry on frozen section of human colon

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  Image collected and cropped by CiteAb under a CC-BY license from the publication

  Western blot - Anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker (ab9023)

  Cytokeratin 8 western blot using anti-Cytokeratin 8 antibody [M20] - Cytoskeleton Marker ab9023. Publication image and figure legend from Belvedere, R., Bizzarro, V., et al., 2016, Sci Rep, PubMed 27412958.

  
  

  ab9023 was used in this publication in western blot. This may not be the same as the application(s) guaranteed by Abcam. For a full list of applications guaranteed by Abcam for ab9023 please see the product overview.

  (A) Cell surface expression of CD44 was analyzed by flow cytometry. The violet area in the plot is relative to human IgG1; CD44 signals are showed in green for WT MIA PaCa-2, in pink for PGS MIA PaCa-2 and in black for ANXA1 KO MIA PaCa-2. (B) RT-PCR for K18 mRNA expression measured on levels of HPRT. Values are expressed using the delta-delta Ct method to derive relative fold change. ***p < 0.001. (C,D) Western blots showing K8, ANXA1, lamin A/C, vimentin and GAPDH expression in WT, PGS and ANXA1 KO MIA PaCa-2 cells. (E,F) Vimentin and lamin A/C relative expression was analyzed by densitometry. The optical density of the protein bands was normalized on GAPDH levels giving to the control band an arbitrary value of 100. The blots were exposed to Las4000 (GE Healthcare Life Sciences) and the relative intensities of bands were determined using ImageQuant software (GE Healthcare Life Sciences). (G) Immunofluorescence analysis to detect ANXA1 (red; panels a, b, c), vimentin (yellow; panels d, e, f), lamin A/C (purple; panel g, h, i) and F-actin (green; panels l, m, n) in WT, PGS and ANXA1 KO MIA PaCa-2. Nuclei were stained with DAPI. Magnification 63 × 1.4 NA. Bar = 10 μm. (H) Fluorescence intensity for ANXA1, vimentin and lamin A/C signals (arbitrary units, A.U.) using ImageJ software; determined on 150 cells (for three independent experiments). The results relative to ANXA1 KO MIA PaCa-2 are representative ± SEM of almost three analyzed clones with a similar behaviour.