Cytokeratin 1 Antibody (LHK1) - BSA Free

Immunocytochemistry Protocol

Culture cells to appropriate density on suitable glass coverslips in 35 mm culture dishes or 6-well plates.

1. Remove culture medium and add 10% formalin to the dish. Fix at room temperature for 5-10 minutes.
2. Remove the formalin and add 0.5% Triton-X 100 in TBS to permeabilize the cells. Incubate for 5-10 minutes.
3. Remove the permeabilization buffer and add wash buffer (i.e. PBS or PBS with 0.1% Tween-20). Be sure to not let the specimen dry out. Gently wash three times for 10 minutes.
4. Alternatively, cells can be fixed with -20C methanol for 10 min at room temperature. Remove the methanol and rehydrate in PBS for 10 min before proceeding.
5. To block nonspecific antibody binding incubate in 10% normal goat serum for 1 hour at room temperature.
6. Add primary antibody at appropriate dilution and incubate at room temperature for 1 hour or at 4 degrees C overnight.
7. Remove primary antibody and replace with wash buffer. Gently wash three times for 10 minutes.
8. Add secondary antibody at the appropriate dilution. Incubate for 1 hour at room temperature.
9. Remove antibody and replace with wash buffer. Gently wash three times for 10 minutes.
10. Nuclei can be staining with 4',6' diamino phenylindole (DAPI) at 0.1 ug/ml, or coverslips can be directly mounted in media containing DAPI.
11. Cells can now be viewed with a fluorescence microscope.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow proper laboratory procedures for the disposal of formalin.

Western Blot Protocol
1. Perform SDS-PAGE on samples to be analyzed, loading 30 ug of total protein per lane.
2. Transfer proteins to membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain according to standard Ponceau S procedure (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot.
5. Block the membrane using standard blocking buffer for at least 1 hour.
6. Wash the membrane in wash buffer three times for 10 minutes each.
7. Dilute mouse anti-Cytokeratin 1 primary antibody in blocking buffer and incubate 1 hour at room temperature.
8. Wash the membrane in wash buffer three times for 10 minutes each.
9. Apply the diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturers instructions) and incubate 1 hour at room temperature.
10. Wash the blot in wash buffer three times for 10 minutes each (this step can be repeated as required to reduce background).
11. Apply the detection reagent of choice in accordance with the manufacturers instructions.
Note: Tween-20 can be added to the blocking or antibody dilution buffer at a final concentration of 0.05-0.2%.