TITLE: Semi-dry western blot and chemiluminescent detection
AUTHORS: Douglas Adamoski, Sandra Martha Gomes Dias

[STEPS]
SECTION: Sample preparation and SDS-PAGE
4. Prepare your samples and run SDS-PAGE as usual.
SECTION: Semi-dry transfer preparation
5. Dilute Timmons-Dunbar Buffer 1X using ultrapure water. Usually, 50 mL for each mini gel (8.4cm x 7cm) to be transfered are enough.
 
SECTION: Blocking and antibody incubation
13. Immediately transfer the membrane to 3% non-fat dry milk diluted in TBS-T (Tris Buffered Saline with 0.05% Tween 20) and incubate with gently rocking for 1 hour.
60 min
SECTION: Chemiluminescent x-ray film development
25. Mix 1 mL of 'Stable Peroxide Solution' with 1 mL of 'Luminol Enhancer Solution' in a clean container.
 
 
SECTION: Semi-dry transfer
10.  Carefully and removing bubbles with an roller, assembly the following:
Bottom electrode
6 x WypAll X60 buffer-soaked sheets
Pre-wet PVDF membrane
6 x WypAll X60 buffer-soaked sheets
Top electrode
 Follow the picture as an guideline:
 

a

SECTION: Semi-dry transfer preparation
6. Soak 12 WypAll X60 sheets (cutted in gel size) in Timmons-Dunbar Buffer 1X.
 
SECTION: Semi-dry transfer preparation
7. Pre-wet PVDF membrane (cutted in gel size) for 30 seconds in 100% methanol
 
30 s
SECTION: Semi-dry transfer preparation
8. Discard the methanol and overlay membrane with Timmons-Dunbar Buffer 1X
 
 
SECTION: Semi-dry transfer
9. Remove gel from electrophoresis chamber and disassembly the sandwich
 
SECTION: Semi-dry transfer
11. Perform the transfer at 0.325 mA/mm2 for 7 minutes when using 0.75mm mini gels.
 
The following table (From Trans-Blot Turbo Blotting System (Cat #170-4155) manual) is quite usefull:
 
   Protocol Name MW (kDa) Time (min) 2 Mini Gels or 1 Midi Gel 1 Mini Format Gel   0.75 mm thick gels > 150 10 2.5 A; up to 25 V 1.3 A; up to 25 V   Mixed 7   < 30 5   1.5 mm thick gels Any 10   
 
SECTION: Semi-dry transfer
12. Disassembly the sandwich and proceed with blocking and antibody incubation.
 
SECTION: Blocking and antibody incubation
15. Pour off washing solution and overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 2/3)
 
5 min
SECTION: Blocking and antibody incubation
17. Pour off washing solution and overlay with desired antibody. Incubate from 1 hour (room temperature) up to 16 hours (4°C) with gently rocking.
4 10
 
SECTION: Blocking and antibody incubation
14. Pour off blocking solution and overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 1/3)
 
5 min
SECTION: Blocking and antibody incubation
16. Pour off washing solution and overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 3/3)
5 min
SECTION: Blocking and antibody incubation
18. Collect antibody and store at 4°C for further uses. Overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 1/3).
5 min
SECTION: Blocking and antibody incubation
20. Pour off washing solution and overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 3/3).
5 min
SECTION: Blocking and antibody incubation
19. Pour off washing solution and overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 2/3).
5 min
SECTION: Chemiluminescent x-ray film development
26. Gently transfer the membrane to this container using tweezers and rock for a few seconds.
 
SECTION: Chemiluminescent x-ray film development
27. Transfer the wet membrane to the X-ray cassete. The best approach is 'sandwich' it with two transparent hard plastic foils (as picture bellow).
 

a

 
SECTION: Chemiluminescent x-ray film development
28. Inside the dark room (remember to left your bright-screen cellphone outside), add one X-ray film over the assembled sandwich and expose for 1 minute.
 
SECTION: Chemiluminescent x-ray film development
29. Remove the film from cassete and put in a container with diluted autoradiography developer solution and incubate for 30 seconds.
30 s
 
SECTION: Chemiluminescent x-ray film development
30. Rinse with tap water
SECTION: Chemiluminescent x-ray film development
31. Put in a container with diluted autoradiography fixer solution and incubate for 1 minute
1 min
 
SECTION: Chemiluminescent x-ray film development
32. Rinse with tap water and let it dry
   
SECTION: Chemiluminescent x-ray film development
33. Overlay the dried film over the original cassete sandwich to tick the prestained marker, stick to blank sheet of paper and scan it.
SECTION: Buffers preparation
1. Transfer buffer: Alcohol-free Timmons-Dunbar
 
Weight reagents as indicated:
30.285 g of Tris-Base
14.41 g of Glycine
Transfer the reagents to a beaker, add 150 mL of ultrapure water, and stir using amagnetic bar until complete dissolution. Using a volumetric flask, complete the volume to 250 mL with ultrapure water. Store at room temperature.

Please cite: Timmons TM, Dunbar BS. Protein blotting and immunodetection. Methods Enzymol (1990), 182:679-688. DOI: 10.1016/0076-6879(90)82053-5

 
   Timmons-Dunbar 4X Transfer Buffer   Reagent Concentration Amount   Tris-Base 1000 mM 30.285 g   Glycine 768 mM 14.41 g   Ultrapure water q.s. 250 mL   
 
 
SECTION: Blocking and antibody incubation
21. Pour off washing soution and overlay with horseradish peroxidase conjugated antibody. Incubate from 1-3 hours at room temperature with gently rocking.
 
SECTION: Blocking and antibody incubation
22. Pour off the secondary antibody. Overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 1/3).
5 min
SECTION: Blocking and antibody incubation
23. Pour off washing solution and overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 3/3).
5 min
SECTION: Blocking and antibody incubation
24. Pour off washing solution and overlay membrane with TBS-T, let it gently rocking for 5 minutes (wash 3/3).
5 min
SECTION: Buffers preparation
2. General Washing Buffer: Tris-buffered saline (TBS) 10X
 
 Weight reagents as indicated:
24,2 g of Tris-Base
87,6 g of Sodium chloride (NaCl)
Transfer the reagents to a beaker, add 900 mL of ultrapure water, and stir using an magnetic bar until complete dissolution. Using a volumetric flask, complete the volume to 250 mL with ultrapure water. Store at room temperature.
SECTION: Gradient PAGE Gel preparation
3.