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.