Aquaporin-5 Antibody - BSA Free

Aquaporin-5 Antibody:
Immunocytochemistry Protocol

Culture cells to appropriate density 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 10 minutes.
2. Remove the formalin and rinse with PBS.
3. Remove PBS and add permeabilization solution (i.e. PBS+0.05%Triton-X100). Be sure to not let the specimen dry out. Wash three times for 5 minutes with washing solution (i.e. PBS).
4. To block nonspecific antibody binding incubate in 10% normal goat serum for 1 hour at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 1 hour at room temperature to overnight at 4C.
6. Remove primary antibody and replace with washing solution. Wash three times for 5 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, washing three times for 5 minutes. Incubate with DAPI at 2ug/ml for 1 minute.
9. Cells can be viewed directly after washing. The plates can also be stored in PBS containing Azide covered in Parafilm (TM). Cells can also be cover-slipped using Fluoromount, with appropriate sealing.

*The above information is only intended as a guide. The researcher should determine what protocol best meets their needs. Please follow safe laboratory procedures.

Aquaporin-5 Antibody:
Western Blot Protocol

1. Perform SDS-PAGE on samples to be analyzed, loading 10-25 ug of total protein per lane.
2. Transfer proteins to PVDF membrane according to the instructions provided by the manufacturer of the membrane and transfer apparatus.
3. Stain the membrane with Ponceau S (or similar product) to assess transfer success, and mark molecular weight standards where appropriate.
4. Rinse the blot TBS -0.05% Tween 20 (TBST).
5. Block the membrane in 5% Non-fat milk in TBST (blocking buffer) for at least 1 hour.
6. Wash the membrane in TBST three times for 10 minutes each.
7. Dilute anti-Aquaporin-5 primary antibody in blocking buffer and incubate overnight at 4C with gentle rocking.
8. Wash the membrane in TBST three times for 10 minutes each.
9. Incubate the membrane in diluted HRP conjugated secondary antibody in blocking buffer (as per manufacturer's instructions) for 1 hour at room temperature.
10. Wash the blot in TBST 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.