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

Lane 1 : A431 cell lysate
Lane 2 : MCF7 cell lysate
Lane 3 : Wild-type HeLa cell lysate
Lane 4 : KRT8 knockout HeLa cell lysate

Lysates/proteins at 20 µg per lane.

Lanes 1 - 4: Merged signal (red and green). Green - ab9023 observed at 55 kDa. Red - 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 ab255400 (knockout 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 (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 (ab216772) and 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.

 

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

Lane 1 : Lysate prepared from human prostate cancer LNCaP cells
Lane 2 : Lysate prepared from human prostate cancer PC3 cells

Lysates/proteins at 50 µg per lane.

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

Developed using the ECL technique.

Performed under reducing conditions.

Observed band size: 50 kDa why is the actual band size different from the predicted?
Additional bands at: 26 kDa, 65 kDa. We are unsure as to the identity of these extra bands.


Exposure time: 4 minutes

See Abreview

Immunohistochemistry on paraffin section of human colon

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.

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 22ºC. The secondary antibody used was DyLight® 488 goat anti-mouse IgG (H&L) (ab96879) at 1/250 dilution for 30 min at 22ºC. Isotype control antibody (black line) was mouse IgG1 [ICIGG1] (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.

Immunohistochemistry on frozen section of human colon