Western Blot using Anti-PARP Antibodies (NB 100-111, -112, -113) and HL60 Cell Extracts

1. HL60 cell extracts were prepared from exponentially growing cells either induced for apoptosis with etoposide or not. Cells were washed once with PBS, suspended at ~4 X 10X6 cells/ml in sample buffer (62.5 mM Tris/HCl, pH 6.8; 6M urea; 10% glycerol; 2% SDS; 0.00125% bromophenol blue; 5% B-mercaptoethanol), sonicated 15 s and incubated at 65 degrees C, 15 min.

Note 1: The purpose of the urea in the sample buffer and the sonication step in the above protocol is to effectively dissociate PARP/DNA interactions.

Note 2: For preparing PARP electrophoresis samples from tissue see G.M. Shah et al. Anal Biochem. 1995 227 1; F. Simonin et al. Anal Biochem. 1991 195 226.

2. Run SDS-PAGE using 20 ml of control and induced HL60 cell extract.

3. Transfer proteins to nitrocellulose (NC) blotting membrane.

4. Block NC with 5% non-fat dry milk in TBST (50 mM Tris/HCl, pH 7.4; 150 mM NaCl; 0.1% Tween 20) for 1 hr at RT with gentle agitation.

5. Incubate NC with appropriate dilution (see data sheet) of anti-PARP in blocking solution for 2.5 hr at RT.

6. Wash membrane with TBST 3X, 10 min. per wash.

7. Incubate NC with secondary antibody (in Blocking solution) using either anti-mouse or anti-rabbit alkaline phosphatase conjugate, as appropriate, for 1 hr at RT.

8. Wash membrane with TBST 3X, 10 min. per wash. Rinse briefly with TBS (TBST w/o Tween).

9. Incubate with BCIP/NBT color development reagent until bands are desired darkness. Rinse with TBS plus 20 mM EDTA to stop color development.

Note 3: Color development times may vary. These procedures are intended only as a guide. The individual user must determine the optimal concentration of primary antibody and the experimental conditions. No warranty or guarantee of performance using these procedures is made or implied.

Extraction of Cellular PARP by PO4 Buffer

Buffer Preparation:

Stock Buffer: This buffer will be used in the preparation of the experimental buffers. The final composition of this buffer is 100mM Tris-Cl (pH 7.4), 10mM MgSO4-7H2O, 500mM Sucrose.

To make 100 ml of Stock Buffer mix the following: 10.0 ml 1M Tris-Cl (pH 7.4) 1.0 ml 1M MgSO4-7H2O 50.0 ml 1M Sucrose 39.0 ml H2O

Buffer A: This is 0.5X Stock Buffer plus protease inhibitors. PMSF ([Final] = 1mM), Leupeptin ([Final] = 0.5 mg/ml), Pepstatin ([Final] = 0.7 mg/ml) and Antipain ([Final] = 5 mg/ml) are also added. To make 20 ml of Buffer A mix the following: 10.0 ml Stock Buffer 0.1 ml 200 mM PMSF in ethanol 10.0 ml Leupeptin (1 mg/ml in H2O) 14.0 ml Pepstatin (1 mg/ml in MtOH) 5.0 ml Antipain (20 mg/ml in H2O) 9.9 ml H2O

Buffer B: Similar to Buffer A but containing 1% (w/v) NP-40 and additional Antipain. To make 10 ml of Buffer B mix the following: 5.0 ml Stock Buffer 1.0 ml NP-40 (10% stock solution) 0.05 ml 200 mM PMSF 5.0 ml Leupeptin (1 mg/ml in H2O) 7.0 ml Pepstatin (1 mg/ml in MtOH) 25.0 ml Antipain (20 mg/ml in H2O) 3.9 ml H2O

Buffer C: Similar to Buffer A but containing 300 mM K2HPO4 (final pH is ~8.5) and additional Antipain. To make 5 ml of Buffer C mix the following: 2.5 ml Stock Buffer 1.5 ml 1M K2HPO4 25.0 ml 200 mM PMSF 2.5 ml Leupeptin (1 mg/ml in H2O) 3.5 ml Pepstatin (1 mg/ml in MtOH) 12.5 ml Antipain (20 mg/ml in H2O) 0.96 ml H2O

Preparation of Cell Extract:

1) Start with 2-20 X 10X6 cells.

2) Put the plates on ice and aspirate the medium.

3) Rinse in ice cold Buffer A.

4) Permeabilize the cells by extraction with 5 ml of Buffer B; let stand on ice for 20 minutes. Drain buffer and store buffer @ -20 degrees C.

5) Extract PARP in 2 ml of cold Buffer C for 20 minutes on ice. Pipette off the extract. Spin in an eppendorf centrifuge @ 2000 X g for 10 minutes at 4 degrees C. Store supernatant at -20 degrees C until analysis is ready.

6) For increasing yield of PARP, transfer the residual cells from the plate to an eppendorf tube. Extract with 1 ml of buffer C at 4 degrees C for 10 minutes. Spin at 3000 X g for 5 minutes and remove supernatant as PARP extract.

7) An aliquot of extract (from step 5 and/or 6) is mixed with 4 vol. of urea-loading buffer for PARP Western. Note 4: For SDS-PAGE and Western blotting of extracts obtained by the above protocol, we recommend the same UREA loading buffer (62.5 mM Tris/HCl, pH 6.8; 6M urea; 10% glycerol; 2% SDS; 0.00125% bromophenol blue; 5% B-mercaptoethanol), described in the standard denaturing extraction/Western Blot procedure for the PARP antibodies.