ABCE1 Antibody - BSA Free

ABCE1 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.

ABCE1 Antibody:
Immunprecipitation Procedure

1. Add 1ug rabbit IgG to 0.5 ml supernatant from HeLa or HEK293 cell line lysate*
2. Add 20 ul protein G plus/protein A-agarose beads
3. React for 1 hour @ RT in rolling oven or shaker
4. Spin down agarose beads and draw the supernatant
5. Add 1-2 ul of anti-ABCE1 (NB 400-116) to 0.5 ml of the cell lysate supernatant
6. React for 4 hours @ RT in rolling over
7. Add 50 ul protein G plus/protein A-agarose to ABCE1 solution for 1 hour in rolling oven @ RT
8. Spin down agarose beads and washed beads 3x with RIPA buffer (+protease inhibitors)
9. Add 2x 50 ul SDS loading buffer (+DTT) to the beads
10. Heat mixture for 10 minutes @ 85 degrees celcius
11. Spin reaction mixture @ 12K rpm for 5 minutes

* Cell line lysate = 75 cm flask dish culture, nearly confluent / 0.5 ml Pierce mammalian protein extraction buffer + protease inhibitors cocktail (100:1 was added for lysate) / lysate centrifuged 12K rpm for 10 min.

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

ABCE1 Antibody:
Western Blot Procedure

1. Run 20 ul of immunoprecipitated protein on a 10% SDS polyacrylamide gel
2. Transfer protein to Immobilon-P nylon membrane: 60mA for 2 hours
3. Block membrane with 5% non-fat milk in PBS + 0.4% Tween-20 [PBS-T] for 1 hour @ RT or overnight @ 4C
4. Wash membrane with PBS-T, 3x [15 min., 5 min., 5 min.], shaking gently
5. Incubate membrane with 1:500 dilution of anti-ABCE1 (NB 400-116), diluted in PBS-T, for 1 hour @ RT
6. Wash membrane with PBS-T, 3x [15 min., 5 min., 5 min.], shaking gently
7. Incubate membrane with anti-rabbit IgG-HRP secondary, diluted in PBS-T, for 1 hour @ RT
8. Wash membrane with more than 100 ml of PBS-T at least 3x (for an 8x6 cm blot)
9. Detect cross-reacting proteins using a Chemiluminescence Reagent kit: expose ~5 seconds

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