Issue 4
October, 2014

Isolation of drug target protein (3)Histone Deacetylase Inhibitor【Vorinostat】
Inhibitor
Summary
Histone deacetylase (HDAC) is an enzyme that performs deacetylation of the histone whichis the main configuration factors in chromatin structure, and vorinostat (SAHA) is known

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as one of the HDAC inhibitors.
We show the result of experiment here to purify the targets of vorinostat in certain condition.
We immobilized vorinostat amino derivative on FG beads (NHS beads) to perform affinityOHNHO

purification of vorinostat binding proteins.

Vorinostat

As the result, several binding proteins were separated from HeLa cell extracts.
Four types of HDAC were identified by western blotting, and ECHS1 (Enoyl(CoA hydratase, short chain 1)
was identified by mass spectrometry in this experimental condition.

Type and amount of the purified proteins may be changed dependent on the amount of compounds immobilized on FG beads.
Therefore, it may be necessary to examine several conditions.

Experimental Information
Vorinostat does not have available functional group. So, we synthesized an amino-derivativeaminoof vorinostat.
When we synthesize a derivative, we introduce a new functional group and position it in a way that does not alter the activity
activit of the compound.
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HNOH
O

N
H

O

NHS beads

Vorinostat immobilized beads

Vorinostat amino derivativeFig.1 Immobilization of Vorinostat amino derivative on NHS beads

Result(kDa)
1．Several binding proteins were separated using FG beads.
Several proteins were eluted with free vorinostat, and it wasIn-+ + : Immobilized Vorinostat+ + : Vorinostat Elution

200

suggested that these proteins specifically interact with116
97.4

vorinostat.

66

Four types of HDAC were confirmed by western blotting,and ECHS1 was identified by mass spectrometry.

M

45

31

HDAC1
HDAC2
HDAC3
HDAC8

ECHS1

21.5
14.4

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TAMAGAWATAMAGAWA SEIKI
SEIKI CO., LTD.

6.5

http://www.FGbeads.com
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Isolation of drug target protein
aterials and method
MMaterials and method■Materials
1. Vorinostat amino derivative
2. NHS beads
3. HeLa cell extracts (cytosolic fraction) – 3mg/ml
4. Binding &Washing Buffer (20mM HEPES-NaOH(pH7.9), 100mM KCl,1mM MgCl2, 0.2mM CaCl2, 0.2mM EDTA, 10%(v/v) glycerol,0.1% NP-40, 1mM DTT, 0.2mM PMSF)
5. Elution Buffer (0.0625M Tris-HCl (pH6.8), 0.005% BPB, 2% SDS,
10% glycerol, 5% 2-mercapto ethanol)
6. Vorinostat
7. Rabbit polyclonal anti-HDAC1 (from Santa Cruz Biotechnology)
8. Rabbit polyclonal anti-HDAC2 (from Santa Cruz Biotechnology)
9. Rabbit polyclonal anti-HDAC3 (from Santa Cruz Biotechnology)
10. Rabbit polyclonal anti-HDAC8 (from Santa Cruz Biotechnology)
11. Anti-Rabbit IgG, HRP-Linked Whole Ab Sheep (from GE Healthcare)
12. Starting Block™ (TBS) Blocking Buffer (from Thermo scientific)
13. TBS-T buffer (137mM NaCl, 2.68mM KCl, 25mM Tris-HCl, pH7.4,
0.1w/v% Tween-20)■Methods 1 (Immobilization)
1. Apply
0.5mg NHS beads and 0.3mM vorinostat amino derivativein 100ul DMF
0.5mg NHS beads and NO vorinostat amino derivativein 100ul DMF (Negative Control)
2. Reaction
1) Immobilization
70minat r.t.
2) Masking
Inactivate unreacted NHS according to recommended conditions.

beads information■■FGProduct name

NHS beads

Product number

TAS8848N1141

Storage temperature-20℃

Storage buffer

Isopropyl alcohol

Size of beads

190nm ± 20nm

Functional groups

200 - 300nmol/mg

3 (Western blotting)■■Methods
1. Perform SDS-PAGE and transfer the protein to a PVDF membrane.
2. Block the membrane with the blocking buffer for 15min at room
temperature.
3. Dilute the anti-HDAC antibody with the blocking buffer to 1/200.
4. Incubate for 60min at room temperature.
5. Dilute the secondary antibody with the TBS-T buffer to 1/1000.
6. Incubate for 60min at room temperature.
7. Wash the membrane with TBS-T buffer three times.
8. Detect with a chemiluminescence substrate.

■Methods 2 (Affinity purification)
1. Wash
Wash beads with washing buffer 3 times at 4oC (on ice).
2. Add sample solution
Add 1000ul HeLa cell extracts to beads.
3. Reaction
Incubate for 240min at 4oC.
4. WashRemove sample solution.
Wash beads with washing buffer 3 times at 4oC (on ice).
5. Elution
Vorinostat elution : Add 40ul elution buffer and resuspend beads.
Boil for 5min at 98oC and remove beads.
Vorinostat elution : +Add 30ul of 25mM vorinostat in washing buffer and resuspend
beads.
Place the tube on ice for an hour to allow bound proteinsto elute by tapping at intervals.
After spin down, separate magnetically.
Transfer the supernatant to a new clean tube.
Add 10ul of 4×elution buffer to the tube.
Boil for 5min at 98oC.
6. Analyze the samples by SDS-PAGE and silver staining

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SEIKI
CO.,
LTD.
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SEIKI
CO.,
LTD.

4 (MS)■■Methods
1. Excising the bands from the gel.
2. Wash the band pieces, then dry the band pieces.
3. Add 500ul/band of 10mM DTT/25mM NH4HCO3.
4. Incubate for 60min at 56℃.
5. Add 500ul/band of 55mM iodeacetamide/25mM NH4HCO3.
6. Incubate for 60min at room temperature.
7. Wash the band pieces with 200ul of 25mM NH4HCO3 for 10min.
8. Wash the band pieces with 500ul of 25mM CH3CN/NH4HCO3(50:50 v/v) for 10min.
9. Dry the band pieces.
10. Add 10ul/band of 10ug/ml trypsin in 25mM NH4HCO3.
11. Incubate overnight at 37℃.
12. Add 50ul/band of 25mM CH3CN/trifluoroacetic acid/ultrapure
water (50:5:45 v/v).
13. Incubate for 30min at room temperature.
14. Transfer supernatant to a new clean tube.
15. Dry the peptides in the tube.
16. Injection on the MS.

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