Detection of Protein on Western Blots by
Enhanced Chemiluminescence
(Hand-written annotations
found on the original copy are indicated by italics.)
Reagents Required:
Ponceau S Stain For 200 ml
Conc. Ponceau S 20 ml (Sigma: C.N. P7767)
dH2O 180 ml
TBST For 1 L
10 mM Tris, pH 7.5 10 ml 1 M Tris, pH 7.5
150 mM NaCl 30 ml 5 M NaCl
dH2O 960 ml
0.05% Tween 80 0.5 ml
NaAzide Solution For 10 ml
20% NaAzide 2 g NaAzide powder
dH2O Bring vol. up to 10 ml
ECL Kit From Amersham (C.N. RPN 2108)
NFDM Nonfat dried milk from grocery store
Procedure:
Notes: This protocol was developed for use with the ECL kit supplied by Amersham. Volumes are for 2 minigel blots. All incubations are performed at room temperature.
1. Prepare Western Blots of protein by standard methods.
2. Rinse blots briefly with dH2O and then stain for ~5 min with Ponceau S. Rinse with dH2O until red background disappears and protein bands become visible. Photograph stained blots for a permanent record. At this point the blots can be store dry between 2 sheets of Whatman paper.
3. Incubate each blot separately (Tupperware) in 25 ml TBST-5% NFDM for 30 min.
4. Incubate in 15 (10) ml primary antibody solution (200 ml affinity purified anti-Adr1p/ 30 ml TBST-1% NFDM-0.05% NaAzide) for 3 h.
5. Wash each blot 4x with 50 ml TBST-1% NFDM for 10 min each. Blots should be thoroughly washed because NaAzide inhibits horseradish peroxidase (HRP).
1st:
2-3 min
2nd:
5 min
3rd
and 4th: 10 -15 min
6. Incubate with 15 ml (fresh) secondary antibody solution (3 ml anti-rabbit
HRP-conjugated antibody/15 ml TBST-1% NFDM; i.e. 6 ml/30
ml for 2 miniblots) for 1 h. Make solution fresh (without NaAzide bacteria
will grow0.
7. Wash each blot 3x with 50 ml TBST-1% NFDM for 10 min.
8. Prepare detection reagent by mixing 3.5 ml of solution 1 with 3.5 ml of solution 2.
9. Lift each blot and allow excess TBST-1% NFDM to drain to one corner. Touch with corner to a paper towel and then briefly law each blot, protein side up, on the towel. Next, place the blots protein side up on a clean plastic surface.
* See below.
10. Pipette detection reagent evenly onto blots and wait exactly 1 min.
11. Lift each blot and allow excess detection reagent to drain to one corner. Touch this corner to a paper towel and then briefly law each blot, protein side up, on the towel. Immediately place each blot protein side down on clean Saran Wrap.
12. Wrap the blots in Saran Wrap and immediately expose to x-ray film at room temperature. Convenient exposure times are 15 sec, 75 sec, 6 min 15 sec, and 2 h.