- Proteins and Peptides
- Lysates and Cell Lines
1. Gels, Whatman paper, and membranes are soaked in electroblotting buffer (25 mM Tris-HCl; 193 mM glycine; 20% methanol) for 15 minutes prior to transferring
2. Proteins separated on SDS-polyacrylamide gels are transferred onto 0.22 micron nitrocellulose sheets by electroblotting in a Transblot BioRad transfer apparatus in 25 mM Tris, 192 mM Glycine, 20% Methanol at 150 mA (70 V). The transfer is carried out for 1 hour at 4 degrees C.
3. Following protein transfer, the filter is blocked with Blotto [1X TBST (10X TBST = 1.5 M NaCl; 100 mM Tris-HCl, pH 8.0; 0.5% Tween 20; 2% NP-40; 0.2% SDS); 5% Carnation dried milk; 0.02% sodium azide] for 1 hour at room temperature on a rotator.
4. Dilute NB 100-116 (anti-APE/ref-1) in Blotto and incubate with the filter, at 4 degrees C overnight, on a rotator.
5. Wash filter 3 times in 1X TBST (50 mM Tris-HCl, 150 mM NaCl, 0.1% Tween 20, pH 7.5) for 10 minutes at 4 degrees C. Secondary antibody (peroxidase conjugated anti-mouse) is incubated with the blot for 30 minutes at room temperature. Cross-reacting proteins are detected using the Chemiluminescence Western Blotting Kit from Boehringer-Mannheim.
HeLa whole cell extracts (NB800-PC1) were used as a positive control for this antibody.
The description that follows is for cultured cells but can be used for cytospins.
1. Split cells into 3.5 cm culture dishes for growth.
2. Wash cells with 5 ml PBS.
3. Fix cells with approx. 3 ml Histochoice (Amresco) for 30 min (Cryostat tissues for 45 min) or use 10% formalin for 30 minutes.
4. Rinse cells with 5 ml TBS, wipe plate dry leaving a small circle of buffer and cells. Mark with red pencil.
5. Pre-block the cells for 30 min. with 10% goat serum in TBS (200 ul).
6. Aspirate blocking solution and add NB 100-116 (anti-APE/ref-1), dilution in 10% goat serum.
7. Incubate in humidified chamber for 3 hours (overnight for tissue at 4 degrees C).
8. Incubate the cells with 1:100 diluted secondary antibody (anti-mouse IgG made in goat) in 10% goat serum and TBS for 1 hour in humidified chamber.
9. Wash 2 times with 5 ml TBS for 5 min each.
10. Block with ABC solution for 30 min.
11. Wash 2 times with 5 ml TBS for 5 min each.
12. Incubate with DAB solution until signal develops. Place into dH2O. Add coverslip with Aqua-mount. TBS: 50 mM Tris, 150 mM NaCl, pH 7.5, ABC and DAB solutions: Vector laboratories