Technical Data
HDAC5 (HD5, FLJ90614, KIAA0600, NY-CO-9, Histone Deacetylase 5)
Suitable for use in Western Blot. Other applications not tested.

Recommended Dilution:
Western Blot:1-4ug/ul detects HDAC5 from a HeLa nuclear extract. HeLa nuclear extract was resolved by electrophoresis, transferred to nitrocellulose and probed with anti-HDAC5 (1ug/ml). Proteins were visualized using a goat anti-rabbit secondary antibody conjugated to HRP and a chemiluminescence detection system.

Optimal dilutions to be determined by the researcher.

Positive Antigen Control:
HeLa nuclear extract. Add an equal volume of Laemmli reducing sample buffer to 10ul of extract and boil for 5 minutes to reduce the preparation. Load 20ug of reduced lysate per lane for minigels.

Storage and Stability:
May be stored at 4C for short-term only. For long-term storage, store at -20C. Aliquots are stable for at least 12 months at -20C. For maximum recovery of product, centrifuge the original vial after thawing and prior to removing the cap. Further dilutions can be made in assay buffer.

Immunoblot Protocol
1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) on a nuclear extract and transfer the proteins to
nitrocellulose. Wash the blotted nitrocellulose twice with water. 2. Block the blotted nitrocellulose in freshly prepared PBS containing 3% nonfat dry milk (PBS-MLK) for 60 minutes at 20-25C with constant agitation. 3. Incubate the nitrocellulose with 1-4ug/ml of anti-HDAC5, diluted in freshly prepared PBS-MLK overnight with agitation at 4C.
4. Wash the nitrocellulose twice with water. 5. Incubate the nitrocellulose in the secondary reagent of choice (a goat anti-rabbit HRP conjugated IgG, 1:5,000 dilution was used) in PBS-MLK for 1.5 hours at room temperature with agitation. 6. Wash the nitrocellulose with water twice. 7. Wash the nitrocellulose in PBS-0.05% Tween 20 for 3-5 minutes. 8. Rinse the nitrocellulose in 4-5 changes of water.
9. Use detection method of choice (enhanced chemiluminescence was used).
PabIgGAffinity Purified
200ug4C (-20C Glycerol)Blue IceHumanRabbit
KLH-conjugated synthetic peptides corresponding to amino acids 536-545 and 194-206 (CTKTGELPRQP and C-KSKEPTPGGLNHS) of human HDAC5.
Purified by Protein A affinity chromatography.
Supplied as a liquid in 0.1M Tris-glycine, pH 7.4, 0.15M sodium chloride, 0.05% sodium azide before the addition of glycerol to 30%.
Recognizes HDAC5, Mr 160kD. Species Crossreactivity: Human. Weak crossreactivity with rat and mouse.
Intended for research use only. Not for use in human, therapeutic, or diagnostic applications.
Grozinger, C.M., et al., PNAS, 96: 48684873, 1999