Western Analysis of Histone Modifications (Aspergillus nidulans)

Materials and Reagents

1. D-glucose
   
2. NaNO₃
   
3. KCl
   
4. MgSO₄^.7H₂O
   
5. KH₂PO₄
   
6. ZnSO₄^.7H₂O
   
7. H₃BO₃
   
8. MnCl₂^.4H₂O
   
9. FeSO₄^.7H₂O
   
10. CoCl₂^.5H₂O
    
11. CuSO₄^.5H₂O
    
12. (NH₄)₆Mo₇O₂₄^.4H₂O
    
13. EDTA
    
14. Spermine
    
15. Spermidine
    
16. PMSF
    
17. Sorbitol
    
18. Halt Protease Inhibitor Cocktail (Thermo Fisher Scientific, catalog number: PI-78437 )
    
19. NaCl
    
20. KCl
    
21. Tris base
    
22. Glycine
    
23. Methanol
    
24. Bio-Rad protein assay (Bio-Rad Laboratories, catalog number: 500-0006 )
    
25. Miracloth (Calbiochem, catalog number: 475855 )
    
26. Rabbit polyclonal antibody to histone H4 acetyl K5 (1:2,500) (Abcam, catalog number: ab51997 )
    
27. Rabbit polyclonal antibody to histone H4 acetyl K8 (1:2,500) (Abcam, catalog number: ab15823 )
    
28. Rabbit polyclonal antibody to human C-terminus histone H4 antibody (1:2,500) (Abcam, catalog number: ab10158 )
    
29. Mouse monoclonal antibody to TATA Binding Protein (TBP) (1:1,000) (Abcam, catalog number: ab61411 )
    
30. Mouse monoclonal antibody to pan acetyl H4 (1:1,000) (Active Motif, catalog number: 39925 )
    
31. Goat anti-mouse alkaline phosphatase conjugated antibody (1:5,000) (Life Technologies, Gibco^®, catalog number: 13864-012 )
    
32. Goat anti-rabbit alkaline phosphatase conjugated antibody (1:5,000) (Pierce Antibodies, catalog number: 31342 )
    
33. 1-Step NBT/BCIP (Thermo Fisher Scientific, catalog number: 34042 )
34. Glucose Minimal Medium (GMM) (see Recipes)
    
35. 2x Laemmli  buffer (see Recipes)
    
36. Nuclear Isolation buffer (see Recipes)
    
37. Resuspension buffer (see Recipes)
    
38. ST buffer (see Recipes)
    
39. TBS-T (see Recipes)
    
40. Western transfer buffer (see Recipes)
    
41. Media Recipes (see Recipes)
    

Equipment

1. Mortar and pestle
   
2. Bio-Rad Mini-Protean gel electrophoresis system (Bio-Rad Laboratories, catalog number: 170-3940 )
   
3. Semi-Dry Blotter (Bio-Rad Laboratories, catalog number: 170-3940)
   
4. Orbital shaker
   
5. 15 ml Falcon tubes
   
6. 50 ml Falcon tubes
   
7. 30 ml centrifuge tubes
8. Microfuge tubes

Procedure