Sodium Potassium ATPase Beta 1 Antibody (464.8 (also known as 8A)) - BSA Free

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 30 minutes.
2. Remove the formalin and add ice cold methanol. Incubate for 5-10 minutes.
3. Remove methanol and add washing solution (i.e. PBS). Be sure to not let the specimen dry out. Wash three times for 10 minutes.
4. To block nonspecific antibody binding incubate in 10% normal goat serum from 1 hour to overnight at room temperature.
5. Add primary antibody at appropriate dilution and incubate at room temperature from 2 hours to overnight at room temperature.
6. Remove primary antibody and replace with washing solution. Wash three times for 10 minutes.
7. Add secondary antibody at appropriate dilution. Incubate for 1 hour at room temperature.
8. Remove antibody and replace with wash solution, then wash for 10 minutes. Add Hoechst 33258 to wash solution at 1:25,0000 and incubate for 10 minutes. Wash a third time for 10 minutes.
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.

1. Run ~20 ug of rat kidney homogenate on a 7.5% SDS-PAGE gel.
2. Transfer protein to the membrane using a Tris-Glycine/Methanol buffer.
3. Block membrane with TBST/5% NFDM for 30 min. at room temperature (~23-27 degrees C).
4. Wash membrane twice, for 5 minutes each, with TBST.
5. Incubate membrane with 1:2,500 dilution of NB300-147 (anti-Na,K-ATPase), diluted in TBST, for 1 hour at room temperature.
6. Wash membrane once for 15 minutes, then four times for 5 minutes each, with TBST.
7. Incubate membrane with 1:15,000 dilution of goat anti-mouse IgG-HRP [(Pierce) stocked 1:2 in glycerol], diluted in TBST, for 1 hour at room temperature.
8. Wash membrane once for 15 minutes, then four times for 5 minutes each, with TBST.
9. Detect cross-reacting proteins using SuperSignal West Pico Chemiluminescent substrate (Pierce), diluted according to manufacturer's guidelines.

*NOTE: Do not boil the protein samples, as boiling causes aggregation of the Na,K-ATPase. The aggregate band will appear at ~150 kDa on Western Blots.