TITLE: General western blot protocol
AUTHORS: Jailyn Izu

[DESCRIPTION]
General western blot protocol

[STEPS]
SECTION: Sample preparation
1. Prepare appropriate cellular extract for analysis (for example, see Transfecting using FuGENE® 4K Transfection Reagent and cell lysis (M-PER) in a 6-well plate protocol).
SECTION: Sample preparation
2. Measure the protein content of each sample (for example, see Bradford Assay protocol).
SECTION: Run gel
3. Add all to a 1.5 mL microcentrifuge tube:

For loading a 1.0 mm x 10 well, 4-12% Bis-Tris (Invitrogen NuPAGE-SDS) mini gel (Invitrogen, #NP0321BOX):
5 µL4x LDS buffer (final concentration : 1X)
2 µL DTT (final dilution is 1:10)
Amount of total protein for each sample (calculate from Bradford Assay, 5-20 μg is typical)
add H2O to 20 µL (final volume)

For loading a 1.5 mm x 15 well, 4-12% Bis-Tris (Invitrogen NuPAGE-SDS) mini gel (Invitrogen, #NP0323BOX):
2.5 µL 4x LDS buffer (final concentration : 1X)
1 µL DTT (final dilution is 1:10)
Amount of total protein for each sample (calculate from Bradford Assay, 5-20 μg is typical)
add H2O to 10 µL (final volume)





SECTION: Run gel
4. Briefly spin the microcentrifuge tubes using a tabletop micro-centrifuge to collect the sample at the bottom of the tube and then load onto the gel. Add 5-10 μl of Li-Cor Chameleon Duo Pre-stained Protein Ladder to Lane 1 for relative molecular weight markers and sample orientation.

Boil samples for 5 min. at 100°C if needed.
SECTION: Run gel
5. Run gel at 100 volts in 1X MOPS-SDS running buffer for50 min 

SECTION: Run gel
6. Prepare transfer buffer solution at room temperature:

To make 2 L:
200 mL10X Tris-glycine transblot buffer
400 mL 100% methanol
ddH2O to 2 L

SECTION: Run gel
7. Pre-wet PVDF membrane for 30 s with 100% methanol, then soak in transfer buffer for about 1 hour.

SECTION: Run gel
8. Soak transfer sponges and Whatman filter paper in transfer buffer for about 1 hour.
SECTION: Transfer
9. Assemble the transfer apparatus:

From bottom to top:
sponge
Whatman paper
gel
PVDF membrane
Whatman paper
sponge
SECTION: Transfer
10. Transfer proteins to the membrane at 40 volts for 120 min 

SECTION: Total Protein Stain
11. After transferring, completely try the membrane by placing it on a piece of Whatman filter paper and incubating in 37 °C for 10 min  

SECTION: Total Protein Stain
12. Stain for total protein by following the protocol for the  
SECTION: Primary antibodies
14. Decant the blocking buffer and add the appropriate primary antibody dilution.
SECTION: Primary antibodies
15. Incubate the membrane and the primary antibody dilution at 4°C overnight with gentle shaking.
SECTION: Secondary antibodies
17. Rinse the membrane briefly with ddH2O. Add secondary antibodies (1:10000) diluted in Licor blocking buffer solution + Tween20 (0.5%) and incubate for 1 hour covered in aluminum foil at room temperature with gentle agitation.60 min 

SECTION: Secondary antibodies
18. After the secondary antibody incubation, wash as outlined in Step 16. Add a fourth and final wash with ddH2O for 15 minutes.60 min 

SECTION: Secondary antibodies
19. Image the membrane using the Li-COR Odyssey M system.
SECTION: Total Protein Stain
13. Block the membranes for 1 hr using Li-Cor Intercept (TBS) blocking buffer    (enough to cover the membrane).60 min 

SECTION: Secondary antibodies
16. Decant the primary antibodies. Wash the membrane with 1X TBS + 0.5% Tween20 + 0.3% sodium azide for 15 minutes with gentle shaking. Repeat the wash 2 more times. 45 min