TITLE: Immunoblotting using precast gels
AUTHORS: Francisco Bustos

[DESCRIPTION]
Immunoblotting is a key technique to visualize changes in protein levels upon treatments. This technique can be challenging and established procedures are required to ensure reproducibility. Here we present our optimized protocol for immunoblotting of protein samples using precast gels and semiwet transfer. This protocol can be used to analyze samples from cell extracts and from in vitro reactions. 

[STEPS]
SECTION: SDS-PAGE Electrophoresis
7. Prepare 500 mL 1X MES-SDS Buffer (50 millimolar (mM) MES, 50 millimolar (mM) Tris Base, 0.1 Mass / % volume SDS, 1 Mass / % volume EDTA, pH 7.3) by diluting    stock 1:20 in Milli-Q water.

SECTION: SDS-PAGE Electrophoresis
8. Unpack a   and remove protective tape and comb.

SECTION: SDS-PAGE Electrophoresis
9. Assemble gel running tank     by placing gel in the tank in front of the buffer core and a second gel or a buffer dam behind the
buffer core. Lock the gel tension wedge in place. Add 1X MES-SDS buffer between the gels and outside the buffer core.

SECTION: SDS-PAGE Electrophoresis
10. Load 8 µL protein molecular weight marker   . If needed load 2 µL  at the end of the gel.

SECTION: SDS-PAGE Electrophoresis
11. Load samples to a maximum volume of 20 µL.

SECTION: SDS-PAGE Electrophoresis
12. Place gel tank lid and connect to power supply   matching colors (for positive and negative).

SECTION: SDS-PAGE Electrophoresis
13. Run gel at 150 V for 120 min at Room temperature or as needed depending on the protein molecular weight and desired separation.

SECTION: Sample preparation
2. In a 1.5 mL microcentrifuge tube add the following reagents in order:  LDS sample buffer/Reducing Agent mix, IPMS lysis buffer, protein sample. For example.


 


SECTION: Sample preparation
1. Mix  and  in a 7:3 ratio (for example: 70 µL LDS sample buffer and 30 µL of Reducing Agent).

SECTION: Sample preparation
3. Poke hole in top of 1.5 mL microcentrifuge tube with a hypodermic needle.
SECTION: Sample preparation
4. Boil samples at 95 °C for 5 min using a ThermoMixer F1.5.
 

SECTION: Sample preparation
5. Centrifuge for 10 s in a Mini Centrifuge at Room temperature 
 

SECTION: Transfer
15. Prepare 2.5 L of 1X transfer buffer (48 millimolar (mM) Tris, 39 millimolar (mM) Glycine, 20 % (v/v) Methanol) by mixing 212 mL 12X Transfer buffer with  1788 mL milli-Q water and 500 mL Methanol .

SECTION: Transfer
16. Cut   or  membrane to the required gel size (usually 9x7 cm) and activate PVDF membrane by incubating in methanol for at least 1 min.
 


SECTION: Transfer
17. Cut   to the required gel size (usually 9x7 cm).

SECTION: Transfer
18. Prepare   . If dirty, boil in warm tap water in the microwave for 5 min. Soak sponge pads in 1X transfer buffer in a plastic tray.

SECTION: Transfer
19. Remove the gel from the cassette by separating the cassette plates with a  . Using the gel knife, cut off the bottom part and wells of the gel as needed.

SECTION: Transfer
20. Place gel in a plastic tray with 1X transfer buffer.
SECTION: Transfer
21. Make transfer sandwich inside the cathode core of the blot module in the following order:
2 sponge Pad 
1 filter paper + gel (grab this from transfer buffer tray), Remove air bubbles by carefully rolling a   on top of the gel.
1 presoaked PVDF or Nitrocellulose membrane. Remove air bubbles using the Blot Roller.
1 filter paper. Remove air bubbles using the Blot Roller.
Sponges up to fill cavity (6-7 total)

SECTION: Transfer
22. Close blot module with the anode core and place this in the buffer chamber. Lock the blot module using the gel tension wedge. 
SECTION: Transfer
23. Place lid  and connect to power supply   matching colors (for positive and negative).
SECTION: Transfer
24. Run gel at 35 V for 90 min at Room temperature .

SECTION: Transfer
25. Unlock blot module by releasing the tension wedge and remove from buffer chamber. Remove membrane from the sandwich and cut excess membrane using scissors. 
SECTION: Transfer
26. Incubate membrane in   in a plastic tray for 5 min with shaking in a rocking shaker.
 

SECTION: Transfer
27. Wash with milli-Q water 3 times, quick washes. Image in Chemidoc MP if needed.
 

SECTION: Blocking
28. Make 1X TBS-T (Tris buffer saline-Tween 20: 20 millimolar (mM) Tris-HCl pH=7.4, 100 millimolar (mM) NaCl, 0.1 % (v/v) Tween-20).
For 1L:
20 mL 1 Molarity (M) Tris pH=7.4
20 mL 5 Molarity (M) NaCl
1 mL Tween-20   
Milli-Q water up to 1 L

SECTION: Blocking
29. Make Blocking solution: 1X TBS-T 5% skimmed milk:
12.5 g powder skimmed milk   .
Fill up to 250 mL with 1X TBS-T.

SECTION: Blocking
30. Add Blocking solution to a Western Blot box    and place the membrane in this solution. Incubate in a rocking shaker for at least 60 min at Room temperature 

SECTION: Primary antibody
31. Prepare antibody in 1X blocking solution (for HRP detection) or in TBST 5 Mass / % volume BSA    (for infrared detection) at the desired concentration.

SECTION: Primary antibody
32. Discard blocking solution from Western blot box and replace by diluted antibody. Incubate at 4 °C 60 min in rocking shaker.

SECTION: Primary antibody
33. Next day recover antibody in tube and wash membrane 3 times for 5 min with 1X TBS-T

SECTION: Secondary antibody
34. Prepare HRP or fluorescently labelled secondary antibody in blocking solution or TBST respectively. 
SECTION: Secondary antibody
35. Add diluted secondary antibody to Western blot box a incubate at Room temperature  for 60 min in rocking shaker.

SECTION: Secondary antibody
36. Wash membrane 4 times for 5 min with 1X TBS-T

SECTION: Secondary antibody
37. Image in Chemidoc MP for HRP detection or LI-COR Odyssey FC imager for infrared detection.
 

 

SECTION: Stripping
39. Prepare Stripping buffer:
weight 15 g glycine   
Dissolve in 800 mL milli-Q water
Adjust pH to 2.2
add 1 g SDS   
10 mL Tween 20    
Bring volume up to 1 L with milli-Q water
SECTION: Stripping
40. Incubate membrane  10 min with stripping buffer  in rocking shaker at Room temperature. 

SECTION: Stripping
41. Repeat step 37
SECTION: Stripping
42. Wash twice with TBST
SECTION: Stripping
43.   and probe with a different antibody.

SECTION: Sample preparation
6. Samples can be stored at -20 °C until the day of running SDS-PAGE electrophoresis.

SECTION: Secondary antibody
38. Membrane can be stored up to a week at 4 °C until performing stripping.

SECTION: Transfer
14. Prepare 12X transfer buffer:
58 g Tris base   
190 glycine   
Milli-Q water up to 2 L