Anti-Cytokeratin 8 KRT8 Antibody Picoband® (monoclonal, 3G9)

1. Flow Cytometry analysis of A549 cells using anti-Cytokeratin 8 antibody (M01421-3).
Overlay histogram showing A549 cells stained with M01421-3 (Blue line). To facilitate intracellular staining, cells were fixed with 4% paraformaldehyde and permeabilized with permeabilization buffer. The cells were blocked with 10% normal goat serum. And then incubated with rabbit anti-Cytokeratin 8 Antibody (M01421-3, 1μg/1x10⁶ cells) for 30 min at 20°C. DyLight®488 conjugated goat anti-rabbit IgG (BA1127, 5-10μg/1x10⁶ cells) was used as secondary antibody for 30 minutes at 20°C. Isotype control antibody (Green line) was rabbit IgG (1μg/1x10⁶) used under the same conditions. Unlabelled sample (Red line) was also used as a control.

Figure 2. Western blot analysis of Cytokeratin 8 using anti-Cytokeratin 8 antibody (M01421-3).
Electrophoresis was performed on a 5-20% SDS-PAGE gel at 70V (Stacking gel) / 90V (Resolving gel) for 2-3 hours. The sample well of each lane was loaded with 50ug of sample under reducing conditions.
Lane 1: human placenta tissue lysates;
Lane 2: human Caco-2 whole cell lysates;
Lane 3: human A549 whole cell lysates;
Lane 4: human A431 whole cell lysates;
Lane 5: human Hela whole cell lysates;
Lane 6: human K562 whole cell lysates;
Lane 7: human HEK293 whole cell lysates.
After Electrophoresis, proteins were transferred to a Nitrocellulose membrane at 150mA for 50-90 minutes. Blocked the membrane with 5% Non-fat Milk/ TBS for 1.5 hour at RT. The membrane was incubated with mouse anti-Cytokeratin 8 antigen affinity purified monoclonal antibody (Catalog # M01421-3) at 0.5 μg/mL overnight at 4°C, then washed with TBS-0.1%Tween 3 times with 5 minutes each and probed with a goat anti-mouse IgG-HRP secondary antibody at a dilution of 1:10000 for 1.5 hour at RT. The signal is developed using an Enhanced Chemiluminescent detection (ECL) kit (Catalog # EK1001) with Tanon 5200 system. A specific band was detected for Cytokeratin 8 at approximately 54KD. The expected band size for Cytokeratin 8 is at 54KD.

Figure 3. IHC analysis of Cytokeratin 8 using anti-Cytokeratin 8 antibody (M01421-3).
Cytokeratin 8 was detected in paraffin-embedded section of human rectal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 1μg/ml mouse anti-Cytokeratin 8 Antibody (M01421-3) overnight at 4°C. Peroxidase Conjugated Goat Anti-Mouse IgG was used as secondary antibody and incubated for 30 minutes at 37°C. The tissue section was developed using HRP Conjugated Mouse IgG Super Vision Assay Kit (Catalog # SV0001) with DAB as the chromogen.

Figure 4. IF analysis of Cytokeratin 8 using anti-Cytokeratin 8 antibody (M01421-3).
Cytokeratin 8 was detected in paraffin-embedded section of human intestinal cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/mL mouse anti-Cytokeratin 8 Antibody (M01421-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Figure 5. IF analysis of Cytokeratin 8 using anti-Cytokeratin 8 antibody (M01421-3).
Cytokeratin 8 was detected in paraffin-embedded section of human mammary cancer tissue. Heat mediated antigen retrieval was performed in EDTA buffer (pH8.0, epitope retrieval solution). The tissue section was blocked with 10% goat serum. The tissue section was then incubated with 2μg/mL mouse anti-Cytokeratin 8 Antibody (M01421-3) overnight at 4°C. DyLight®488 Conjugated Goat Anti-Mouse IgG (BA1126) was used as secondary antibody at 1:100 dilution and incubated for 30 minutes at 37°C. The section was counterstained with DAPI. Visualize using a fluorescence microscope and filter sets appropriate for the label used.

Figure 6. IF analysis of Cytokeratin 8 using anti-Cytokeratin 8 antibody (M01421-3).
Cytokeratin 8 was detected in immunocytochemical section of U20S cell. Enzyme antigen retrieval was performed using IHC enzyme antigen retrieval reagent (AR0022) for 15 mins. The cells were blocked with 10% goat serum. And then incubated with 2μg/ml mouse anti-Cytokeratin 8 Antibody (M01421-3) overnight at 4°C. Biotin Conjugated Goat anti-Mouse IgG (BA1001) was used as secondary antibody and incubated for 30 minutes at 37°C. The section was developed using Cy3 Conjugated Avidin (BA1037). Visualize using a fluorescence microscope and filter sets appropriate for the label used.