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

1. Whole-cell extract preparation
   1. Inoculate a single colony in 5 ml of YPD medium in a 14 ml Snap-cap tube and grow overnight at 30 °C shaking with 220 rpm.
      
   2. Dilute overnight culture to an OD₆₀₀ of 0.1 in 5 ml YPD medium in a 14 ml Snap-cap tube and grow cells to OD₆₀₀ of 1 at 30 °C shaking with 220 rpm.
      Note: For strains/conditions with different cell morphologies determine cell number by CASY measurement according to the CASY operator manual and use a total number of 5 x 10⁷ cells. Flasks can also be used instead of Snap-cap tubes.
      
   3. Harvest cells by centrifugation in 15 ml Falcon tubes for 3 min at 1,500 x g.
      
   4. Resuspend pellet in 1 ml ice-cold H₂O.
      Note: Work on ice from now on.
      
   5. Transfer suspension to 1.5 ml tube.
      
   6. Add 150 μl ice-cold Yex lysis buffer, vortex thoroughly and incubate on ice for 10 min.
      
   7. Add 150 μl ice-cold 50% (w/v) TCA and incubate on ice for 10 min to precipitate proteins.
      
   8. Spin for 5 min at 10,000 x g 4 °C and discard supernatant with a pipette.
      
   9. Spin for 1 min at 10,000 x g 4 °C and remove rest of the supernatant.
      
   10. Resuspend pellet in 100 μl protein sample buffer.
       Note: Resuspend by pipetting; if pH turns acidic (protein sample buffer turns yellow), add 1 M Tris base to increase pH (until sample buffer turns blue again).
       
   11. Incubate at 37 °C for 15 min shaking at 900 rpm.
       
   12. Spin down cell debris at 10,000 x g for 5 min and use 10 μl (0.5 OD₆₀₀ units) of the supernatant for SDS-PAGE.
       
       
2. SDS-PAGE and western blotting
   1. Load samples on a polyacrylamid gel (4.5% stacking gel and 20% running gel).
      
   2. Load 0.5 μg recombinant histone H4 in protein sample buffer as negative control and 2 μg calf histones in protein sample buffer as positive control for acetylation analysis.
      Note: Gel cast as described previously (Sambrook and Russell, 2001); Bio-Rad Mini-Protean gel electrophoresis system was used.
      
   3. Run gel at 150 V.
      
   4. Transfer proteins to nitrocellulose membrane by electroblotting using the Bio-Rad Mini Trans-Blot cell.
      Assemble blotting cassette in transfer buffer according to the Mini Trans-Blot instruction manual and run at 200 mA for 1 h at room temperature.
      
   5. Block membrane for 1 h at room temperature on an orbital shaker using 5% (w/v) BSA in TBS.
      
   6. Incubate with primary antibody diluted in TBS-T overnight at 4 °C on an orbital shaker.
      
   7. Pour off primary antibody solution and wash 3 x 10 min in TBS-T at room temperature.
      Note: Primary antibody solution can be reused several times (depending on the antibody); if solution is reused, add 0.033% sodium azide as preservative.
      
   8. Incubate with secondary antibody diluted 1:10,000 in TBS-T for 45 min at room temperature on an orbital shaker.
      Note: IRDye 800CW or IRDye 680RD secondary antibodies can be used.
      
   9. Pour off secondary antibody solution and wash 3 x 10 min in TBS-T at room temperature.
      
   10. Rinse blot briefly with PBS and scan using the LI-COR Odyssey CLx Infrared scanner.
       Note: Scanner settings: Intensity: 5; Resolution: 168 μm; Quality: medium.
       

Recipes

1. YPD medium
   10 g/L Bacto Yeast Extract
   20 g/L Bacto Peptone
   20 g/L Glucose (add after autoclaving as 10x stock)
   
2. Yex lysis buffer
   1.85 M NaOH
   7.5% (v/v) β-Mercaptoethanol (freshly added)
   
3. Protein sample buffer
   40 mM Tris-HCl, pH 6.8
   8 M Urea
   5% (w/v) SDS
   0.1 mM EDTA
   1% (v/v) β-Mercaptoethanol (freshly added)
   0.1 g/L Bromphenolblue
   
4. Running buffer
   25 mM Tris
   192 mM Glycine
   0.1% (w/v) SDS
   
5. Transfer buffer
   25 mM Tris
   192 mM Glycine
   20% (v/v) Methanol
   
6. TBS
   25 mM Tris
   140 mM NaCl
   2.5 mM KCl
   pH 7.4
   
7. TBS-T
   TBS with 0.1% (v/v) Tween 20
   
8. PBS
   140 mM NaCl
   2.5 mM KCl
   8.1 mM Na₂HPO₄
   1.5 mM KH₂PO₄
   pH 7.3
   

Acknowledgments

We thank all laboratory members for helpful discussions. This work was supported by a grant from the Christian Doppler Society, and in part by a grant from the Austrian Science Foundation (Project FWF-P-25333), to K.K. M.T. was supported through the Vienna Biocenter PhD Programme WK001.

References

1. Sambrook, J. & Russell, D.W. (2001). Molecular Cloning, Volume 3, 3^rd edition, Cold Spring Harbor Laboratory Press.
   
2. Tscherner, M., Stappler, E., Hnisz, D. and Kuchler, K. (2012). The histone acetyltransferase Hat1 facilitates DNA damage repair and morphogenesis in Candida albicans. Mol Microbiol 86(5): 1197-1214.