ABCG8 Antibody - BSA Free

ABCG8 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 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.

ABCG8 Antibody:
Western Blot Protocol

1. Load ~50 micrograms of lysate (ie: liver membrane) onto an 8% SDS-PAGE, reducing.

2. Block the protein transferred membrane for 30 minutes in blocking buffer [5% NFDM, 10 mM Tris (pH 7.6) saline, 0.2% Tween-20] at room temperature.

3. Incubate the membrane in diluted primary anti-ABCG8 [cat# NB 400-117], 1:250 for 1 hour at room temperature.

4. Wash the membrane 3x 5 minutes in blocking buffer without milk.

5. Incubate the membrane in diluted secondary anti-Rabbit IgG conjugated to HRP.

6. Wash the membrane 3x 5 minutes in blocking buffer without milk.

7. Wash the membrane for 5 minutes in TBS.

8. Develop using West-Pico ECL reagent (Pierce).