Western Blot Procedure
1) Cells were pelleted, washed in 1XPBS, suspended in ice water (~ 5 x 10(6) cells/ml), and placed on ice
2) Lysates were prepared with the addition of 2X lysis buffer [2% SDS/ 50mM Tris-HCl / 10% glycerol]
3) Lysates were heated to 95 degrees C for 3 minutes and then microfuged at room temperature for 10 minutes
4) 50 ug of lysate were electrophoresed (150 V) through a 4-15% PAGE
5) Proteins were transferred (60 V) onto an Immobilon-P membrane (Millipore Corp.) for 45 minutes
6) The blot was blocked overnight at 4 degrees C in blocking buffer [1XPBS, pH 7 / 5% nonfat milk / 0.1% Tween-20]
7) Washed the blot in 1XPBS / 0.1% Tween-20
8) Incubated the blot with 1 ug/ml of (NB500-201) anti-Survivin antibody, diluted in blocking buffer, for 2 hours at room temperature
9) Washed the blot in 1XPBS / 0.1% Tween-20
10) Reacted the blot with HRP-conjugated donkey anti-rabbit Ig, diluted in 1XPBS / 0.1% Tween-20, for 30 minutes at room temperature
11) Washed the blot in 1XPBS / 0.1% Tween-20
12) Visualized blot by ECL and autoradiography
NOTE: HeLa whole cell extracts (NB800-PC1) were used as a positive control for this antibody.
Immunohistochemistry
1) 5 mm tissue sections were cut and baked on slides overnight at 60 degrees C
2) Sections were deparaffinized in xylene for 5 hours
3) Sections were washed twice in ethanol
4) Endogenous peroxidase activity was quenched with 1.5% hydrogen peroxide / methanol for 10 minutes
5) Washed sections in ethanol and then water
6) Slides were placed in a 4-qt Wear-Ever pressure cooker containing 1.5L of boiling 9mM sodium
citrate, pH 6, for ~ 6 minutes, until the pressure valve was released
7) Slides were gently cooled by filling the cooker with tap water
8) Washed sections 3 times with water and once with 1XPBS, pH 7
9) Staining was either performed using a Histostain-Plus kit (Zymed) with DAB as the chromogen or a Vectastain Elite ABC kit (Vector Laboratories) with AEC as the chromogen
10) Slides were placed over moistened paper towels, in a covered tray
11) (NB500-201) Anti-Survivin antibody was diluted in 1XPBS, pH 7 / 0.5% BSA / 5% normal goat serum to a concentration of 0.5 ug/ml
12) Diluted primary antibody was applied to the sections and incubated overnight at 4 degrees C
Immunoprecipitation Procedure
1) Lyse cells plated in a 60mm dish:
a) 300 ul CHAPS buffer [50mM Tris-HCl, pH 7.5/50mM NaCl/1mM EDTA/1% NP-40/0.1% CHAPS/1mM NaVO4/1mM PMSF]
b) Rock for 20 minutes at 4 degrees C
2) Harvest lysate and spin down the insoluble material at 14K rpm
3) Collect soluble fraction
4) Pre-clear lysate with 40 ul of 50:50 slurry of Protein A beads, rocking for 1 hour at 4 degrees C
5) Spin down beads at 2K rpm, at 4 degrees C
6) Collect pre-cleared lysate
7) Incubate lysate with 5-7ug of anti-Survivin (NB 500-201) overnight, rocking at 4 degrees C
8) Add 50 ul of Protein A 50:50 slurry for 2 hours, rocking at 4C
9) Wash beads with 200 ul of CHAPS buffer, three times
10) Denature immune complex by adding 2x Sample Buffer, containing 2-ME
11) Boil for 10 minutes and load onto an SDS-gel.