Immunoblot Analysis of Histone H4 Acetylation and Histone H2A Phosphorylation in Candida albicans

Materials and Reagents

1. Candida albicans
   
2. TCA (trichloroacetic acid) (Merck KGaA, catalog number: 641730 )
   
3. Recombinant histone H4 (New England Biolabs, catalog number: M2504S )
   
4. Calf histones (Sigma-Aldrich, catalog number: H9250 )
   
5. 0.033% sodium azide (Merck KGaA, catalog number: 8223350250 )
   
6. Nitrocellulose membrane (Millipore, catalog number: Protran BA79)
   
7. Whatman filter paper 3 MM Chr (Whatman, catalog number: 3030-917 )
   
8. BSA (PAA Laboratories GmbH, catalog number: K41-001 )
   
9. Sodium azide (Merck KGaA, catalog number: 822335 )
   
10. Rabbit polyclonal antibody to histone H4 acetyl K5 (Abcam, catalog number: ab51997 ) (dilution: 1:5,000)
    
11. Rabbit polyclonal antibody to histone H4 acetyl K8 (Active Motif, catalog number: 39172 ) (dilution: 1:3,000)
    
12. Rabbit polyclonal antibody to histone H4 acetyl K12 (Millipore, catalog number: 07-959 ) (dilution: 1:3,000)
    
13. Rabbit polyclonal antibody to histone H4 C-terminus (Abcam, catalog number: ab10158 ) (dilution: 1:1,000)
    
14. Rabbit polyclonal antibody to histone H2A phospho-serine 129 (Active Motif, catalog number: 39271 ) (dilution: 1:2,000)
    
15. Rabbit polyclonal antibody to histone H2A (Active Motif, catalog number: 39236 ) (dilution: 1:5,000)
    
16. IRDye 800CW goat anti-rabbit IgG (H + L) (LI-COR, catalog number: 926-32211 )
    
17. IRDye 680RD goat anti-rabbit IgG (H + L) (LI-COR, catalog number: 926-68071 )
    
18. 4.5% stacking gel
    
19. 20% running gel
    
20. Bacto Yeast Extract (Becton, Dickinson and Company, catalog number: 212720 )
    
21. Bacto Peptone (Becton, Dickinson and Company, catalog number: 211820 )
    
22. Glucose (Merck KGaA, catalog number: 346351 )
    
23. β-Mercaptoethanol (Sigma-Aldrich, catalog number: M3148-100ML )
    
24. Urea (Sigma-Aldrich, catalog number: U5378-1KG )
    
25. SDS (AppliChem GmbH, catalog number: A1112,1000 )
    
26. EDTA (Sigma-Aldrich, catalog number: E5134-1KG )
    
27. Bromphenolblue
    
28. YPD medium (see Recipes)
    
29. Yex lysis buffer (see Recipes)
    
30. Protein sample buffer (see Recipes)
    
31. SDS-PAGE running buffer (see Recipes)
    
32. Running buffer (see Recipes)
    
33. Transfer buffer (see Recipes)
    
34. TBS and TBS-T (see Recipes)
    
35. PBS (see Recipes)
    

Equipment

1. Mini-Protean gel electrophoresis system (Bio-Rad, model: 165-8000 )
   
2. Mini Trans-Blot cell (Bio-Rad, model: 170-3930 )
   
3. 1.5 ml microcentrifuge tubes
   
4. 15 ml Falcon tubes
   
5. 14 ml Snap-cap tubes
   
6. Eppendorf Thermomixer comfort (Eppendorf, model: 5355 000.011 )
   
7. Shaking incubator
   
8. Orbital shaker
   
9. LI-COR Odyssey CLx Infrared scanner (LI-COR, model: P/N 9140-WP )
   
10. CASY Cell Counter Model TT (Roche, catalog number: 05651735001 )
    

Software

1. LI-COR Odyssey CLx imaging system

Procedure