NUT Antibody - BSA Free

NUT Antibody:
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.

NUT Antibody:
Reagents needed:
a. Washing Buffer: Tris Buffer Saline with 0.01% of tween 20).
b. Blocking Buffer: 5% skimmed milk powder in washing buffer).
c. Secondary antibody, Horseradish peroxidase conjugated.
d. Chemiluminescent solution (SuperSignal WestPicoTM, Pierce).

Western blot Method:
1. Perform SDS-PAGE using PVDF membrane. Cut into strips.
2. Activate strips with methanol by dipping them into methanol for 5 min.
3. Discard the methanol and take fresh methanol to repeat step b.
4. Let the strips dry, and then add blocking solution and incubate at RT in a shaker for 30-45 minutes.
5. Dilute primary antibody in blocking buffer. Incubate the number of strips required with the diluted primary antibody at room temperature for 2 hours in a shaker.
6. Wash strips two times with washing buffer at 30 minutes intervals.
7. Dilute HRP conjugated secondary antibody in blocking buffer. Add diluted secondary antibody to the membrane strips and incubate for exactly 1 hour while shaking at RT.
8. Wash the strips with washing buffer for 2-3 hours with 3 to 4 changes on a shaker. This helps in reducing the back ground staining.
9. Prepare the chemiluminescent solution (SuperSignal WestPicoTM) by mixing solution A and Solution B at 1:1. Mix well. Soak the strip in the chemiluminescent solution; keep for 3-5 minutes under constant shaking.
10. Expose the membrane to a sheet of film and develop.