TITLE: Western Blotting
AUTHORS: Eric ECS Cordeiro-Spinetti

[DESCRIPTION]
It's a classic

[STEPS]
SECTION: Before Starting
1. 
 Make RIPA buffer (to be used in 3 months after adding Nuclease)
 
 
     RIPA buffer      100mL         50mM   Tris HCl, PH 7.4      5mL (1M   stock)         150mM   NaCl      5mL (3M   stock)         1%   Triton X-100 or NP-40      1 mL            0.1%   SDS      1mL   (10% supply center solution)         1mM   EDTA       0.2 mL   (0.5M stock)         10mM   Naf      0.042g   (@L5P1 chemical room)         0.5%   Sodium deoxylcholate      0.5g   (@L5P1 chemical room)           Add   ddH2O to 1000ml                      Add   PMSF to a final concentration of 1mM and any other protease inhibitors   immediately before use.                Add 1   uL Pierce™ Universal Nuclease per 1mL    


 
 
 
SECTION: Running, Transfer and Blot
15. Quantify protein
SECTION: Running, Transfer and Blot
15.1. 799 uL water + 1 uL lysate 
+ 200 uL 5x Bradford reagent
SECTION: Running, Transfer and Blot
15.2. Read at 595 nm
SECTION: Running, Transfer and Blot
16. Load 50 µg of protein/well
SECTION: Running, Transfer and Blot
17. Loading buffer 50 µL β-mercaptoethanol + 950 µL 2x Laemli Buffer

SECTION: Running, Transfer and Blot
18. Water up to 15 µL

SECTION: Running, Transfer and Blot
19. Boil at95 °C before loading  15 s

SECTION: Running, Transfer and Blot
20. Add 10 µLof ladder (Precision Plus Protein - Dual Color Standards)

SECTION: Running, Transfer and Blot
21. Run at 120-150 volts 60 min

SECTION: Running, Transfer and Blot
22. Activate PVDF membrane in methanol
SECTION: Running, Transfer and Blot
24. Equilibrate gel in Towbin buffer 10 min

SECTION: Running, Transfer and Blot
23. Soak filter paper in Towbin Buffer
SECTION: Running, Transfer and Blot
25. Semi-dry transfer
SECTION: Running, Transfer and Blot
25.1. 90' (<50KDa) or 120' (>50KDa)
SECTION: Running, Transfer and Blot
25.2. Mount sandwich (paper-membrane-gel-paper)
SECTION: Running, Transfer and Blot
25.3. 45 mA/gel (constant current)  
SECTION: Running, Transfer and Blot
26. Cut out LEFT UPPER corner of the membrane
SECTION: Running, Transfer and Blot
27. Block membrane with 5% BSA in PBS 0.1% Tween (PBST) for 30 min

SECTION: Running, Transfer and Blot
28. Add primary antibody in BSA-PBST and incubate overnight 1080 min at 4 °C

SECTION: Running, Transfer and Blot
29. Wash 3x with PBST for 5 min

SECTION: Running, Transfer and Blot
30. Add HRP secondary antibody in BSA-PBST and incubate for60 minat room temperature on a shaker

SECTION: Running, Transfer and Blot
31. Wash 3x with PBST for5 min

SECTION: Running, Transfer and Blot
32. Add 500 µL ECL (enhanced chemilumescent) per membrane strip
SECTION: Cell lysis
3. Trypsinize cells
SECTION: Cell lysis
4. Wash pellet with PBS
SECTION: Cell lysis
5. Remove all supernatant with micropipette
SECTION: Cell lysis
6. Freeze pellet in liquid N2 
SECTION: Cell lysis
7. Thaw quickly at 37 °C 

SECTION: Cell lysis
8. Resuspend cell pellet on buffer 100 µL

SECTION: Cell lysis
9. Vortex 15 s

SECTION: Cell lysis
10. Incubate on Ice 20 min

SECTION: Cell lysis
12. Collect supernatant
SECTION: Running, Transfer and Blot
14. Collect supernatant without disturbing the pellet
SECTION: Before Starting
2. SDS Running Buffer
SECTION: Cell lysis
11. Vortex 15 s

SECTION: Cell lysis
13. Centrifuge 10000 RPM for 5 min