HDAC2 Immunoprecipitation (IP) and Activity Assay Kit
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HDAC2 Immunoprecipitation (IP) and Activity Assay Kit Catalog Number EPI012 Storage Temperature –20 °C TECHNICAL BULLETIN Product Description AFC Standard (1 mM) (Yellow Cap) 100 mL Histone deacetylases (HDACs) play a central role in Catalog Number EPI012E controlling cell cycle regulation, cell differentiation, and tissue development. These proteins have crucial roles Rabbit HDAC2 Antibody (Red Cap) 250 mL in development and physiology, especially in the heart Catalog Number EPI012F and nervous system. They are also deeply involved in cellular proliferation, cell cycle, and apoptosis. Rabbit IgG (Control Antibody) (Green Cap) 100 mL Catalog Number EPI012G The HDAC2 Immunoprecipitation (IP) and Activity Assay Kit provides an antibody-based method to Protein-A/G SepharoseÒ Beads (Blue Cap) 650 mL specifically immunoprecipitate the HDAC2 complex Catalog Number EPI012H from cells and tissues, and to measure HDAC2 activity fluorometrically. HDAC2 is immunoprecipitated from Positive Control (Jurkat Cell Lysate) (Violet Cap) 1 vl cell, nuclear, or tissue extract(s) using HDAC2 antibody Catalog Number EPI012I followed by capturing the complex on protein-A/G beads. The immunoprecipitated complex is incubated Reagents and equipment required but not provided. with the HDAC substrate. Only the deacetylated · 96 well black plates for fluorometric assays. substrate is cleaved by the Developer to produce a · Fluorescence plate reader fluorophore, which can be easily analyzed using a · Rotary mixer fluorescence plate reader. The kit is suitable for · Phosphate Buffered Saline (Catalog Number measurement of HDAC2 activity of immunoprecipitated P5368 or equivalent) complex or purified enzyme from human, mouse, or rat · Protease Inhibitor Cocktail (Catalog Number P8340 samples. or equivalent) Components · CelLyticä NuCLEARä Extraction Kit (Catalog The kit is sufficient for 25 assays in 96 well plates. Number NXTRACT or equivalent) HDAC Assay Buffer (Wide Mouth Bottle) 9 mL Precautions and Disclaimer Catalog Number EPI012A This product is for R&D use only, not for drug, household, or other uses. Please consult the Safety Cell Lysis Buffer (Narrow Mouth Bottle) 100 mL Data Sheet for information regarding hazards and safe Catalog Number EPI012B handling practices. Storage/Stability HDAC Substrate (Amber Cap) 100 mL Catalog Number EPI012C The kit is shipped on wet ice. Store the kit at –20 °C, protected from light. However, some components have different storage temperatures once reconstituted. See Developer (Orange Cap) 500 mL Preparation Instructions for storage conditions of Catalog Number EPI012D individual components. 2 Preparation Instructions Procedure Briefly centrifuge vials before opening. Use ultrapure Sample Preparation water for the preparation of reagents. To maintain 1. Cell Lysate reagent integrity, avoid repeated freeze/thaw cycles. a. Grow cells in 6 or 12 well plates, treat as Read the entire protocol before performing the assay. desired. b. For adherent cells, remove medium and wash HDAC Assay Buffer – Store at –20 °C or 2–8 °C. Briefly cells with PBS. warm to 37 °C before use. c. For suspension cells, collect cells by centrifugation, wash cells with PBS at room Cell Lysis Buffer – Thaw Cell Lysis Buffer and add temperature, and collect cells again by protease inhibitors as per manufacturer’s centrifugation. instructions. Make fresh as needed and keep on ice d. Remove the PBS, place the plate on ice, and while in use. add cold Lysis Buffer containing protease inhibitors. Add 125 mL/well (12 well plate) or HDAC Substrate – Store at –20 °C. 250 mL/well (6 well plate). e. Keep on ice for one minute. Developer – Aliquot 250 mL into tubes and store at f. Scrape the cells and gently transfer the –20 °C. Keep on ice while in use. Use within disrupted cell suspension into a cooled 2 months. microcentrifuge tube. g. Mix on a rotary mixer at 2–8 °C for 30 minutes. AFC (7-amino-4-trifluoromethyl coumarin) Standard – h. Centrifuge at 10,000 ´ g for 10 minutes at Store at –20 °C. 2–8 °C, then discard cell debris pellet. Protein-A/G Sepharose Beads (50% slurry in 20% 2. Nuclear Extract ethanol aqueous solution) – Store at 2–8 °C after Prepare nuclear extracts from 2 ´ 105 to 2 ´ 106 thawing. Do not freeze. cells using CelLytic NuCLEAR Extraction Kit (Catalog Number NXTRACT) or other equivalent method. Positive Control – Reconstitute with 25 mL of water. Mix gently by pipetting. Aliquot and store at –70 °C. Use 3. Tissue Extract within 2 months. a. Snap freeze dissected tissue and immediately grind to a fine powder using a mortar and Phosphate Buffered Saline (PBS) – Chill PBS before pestle in a liquid nitrogen bath. use. Add protease inhibitors as per manufacturer’s b. Transfer the ground tissue to a pre-weighed instructions. Make fresh as needed and keep on ice chilled tube. while in use. c. Weigh the powder and add 300 mL of Lysis Buffer containing protease inhibitors per 5 mg of tissue. d. Mix on a rocker at 2–8 °C for about an hour. e. Pass the lysate 3 times through a 25 gauge needle. f. Collect the lysate and centrifuge at high speed at 2–8 °C for 5 minutes to remove debris. g. Transfer the supernatant to a fresh tube. Note: Repeated freeze-thaw of sample will cause loss in HDAC activity. 4. Protein Estimation Determine the protein concentration using the Bradford Assay. 3 Immunoprecipitation HDAC Activity Assay 1. Antibody Binding 1. Warm HDAC Assay Buffer to 37 °C prior to use. a. Cell lysates, Nuclear and Tissue extracts Mix enough reagents for the number of assays to (CL/NE/TE) are in the linear range for HDAC be performed including sample and background activity when 50–100 mg protein is used. If control. For each assay, prepare 168 mL of the samples have high HDAC levels, the amount Reaction Mix according to the scheme in Table 1. used needs to be standardized. b. For each IP reaction (sample or control), add Table 1. 50–100 mg CL/NE/TE to a chilled tube on ice. Reaction Mix c. Add 10 mL of HDAC1 Antibody to samples. Add 10 mL of Control Antibody to samples to Reagent Volume prepare background controls. HDAC Assay Buffer 164 mL d. Bring the volume to 500 mL with PBS containing HDAC Substrate 4 mL protease inhibitor. Total volume 168 mL e. Incubate overnight at 2–8 °C on a rotary mixer. 2. Add 168 mL of Reaction Mix to each sample and 2. Preparation of Protein-A/G Beads: background control tube, mix gently, and incubate a. Use wide bore pipette tips when pipetting at 37 °C for two hours. beads. b. Wash the protein-A/G beads (25 mL of 50% 3. The Positive Control is used to measure total slurry per reaction) 2 times with 1 mL of PBS, by centrifuging at 14,000 ´ g for 10 seconds HDAC Activity. In a separate tube add 2–5 mL of and aspirating the supernatant between Positive Control, 4 mL HDAC Substrate, and adjust washes. the volume to 180 mL with HDAC Assay Buffer. Mix c. Suspend as 50% slurry in PBS. gently and incubate at 37 °C for two hours. 3. Bead Capture: 4. Add 20 mL of the Developer to each tube and mix. a. After overnight incubation of the sample/ Incubate for 30 minutes at 37 °C. antibody mixture (step 1e), add 25 mL of the protein-A/G bead slurry (step 2c) to each tube 5. Preparation of AFC Standards for Standard Curve– and incubate for an hour at 2–8 °C. Dilute the 1 mM AFC Standard to 10 mM by adding b. Collect the beads by centrifugation at 14,000 ´ g for 10 seconds at 2–8 °C. 10 mL of 1 mM AFC Standard to 990 mL of water. c. Wash beads 3 times with 1 mL of PBS, by Add 0, 20, 40, 60, 80, and 100 mL of diluted 10 mM centrifuging at 14,000 ´ g for 10 seconds and AFC Standard into individual wells in a 96 well aspirating the supernatant between washes. black plate. Adjust the volume to 100 mL/well with d. Assay HDAC Activity immediately. HDAC Assay buffer to generate 0, 200, 400, 600, 800, and 1,000 pmole/well of AFC Standard, respectively. Mix well. 6. Centrifuge the Positive Control, Background Control, and Sample tubes at 14,000 ´ g for 2 minutes at room temperature. 7. Transfer 100 mL of each reaction supernatant to individual wells in a black plate. 8. Read fluorescence at (lex = 380/lem = 500 nm). 4 Results HDAC Activity Calculations Subtract the Background Control reading from all Plot the AFC Standard Curve from the fluorescence Sample readings. Apply the corrected sample reading measurements of the AFC Standards. to the AFC Standard Curve to obtain B (pmole of AFC Note: A new standard curve must be set up each time in the sample wells). the assay is run. HDAC Activity = 2 ´ B/TS = pmole/min/mg = milliunits Figure 1. AFC Standard Curve. where: B = AFC amount from the Standard Curve (pmole) 2 = Sample dilution factor* T = Reaction time (120 minutes) S = Sample amount (mg) * Reaction Volume (Sample and Background Control) = 200 mL = ~12 mL beads + 168 mL reaction mix + 20 mL Developer. 100 mL of the reaction is used to measure the fluorescence and hence the sample dilution factor is 2. Unit definition: One unit of HDAC is the amount of enzyme that generates one nanomole of deacetylated substrate/min/mg at 37 °C. Figure 2. HDAC2 IP Activity Assay of HeLa cell lysate and Nuclear Extract and HDAC Activity Assay of Positive Control. Assays were performed according to the kit protocol. CelLytic and NuCLEAR are trademarks of Sigma- Aldrich Co. LLC. Sepharose is a registered trademark of GE Healthcare.