Data Sheet Histone H3 Dimethyl-K27 ELISA Detection Kit Catalog # 53030

Transcription

1 Data Sheet Histone H3 Dimethyl-K27 ELISA Detection Kit Catalog # DESCRIPTION: The Histone H3 Dimethyl-K27 Detection Kit is designed to detect dimethylation of lysine 27 of Histone H3 (H3K27me2) in cell lysates. H3K27 is the substrate of the EZH2 methyltransferase complex. Abnormal methylation of H3K27 is associated with a number of cancers, including breast, colorectal, and Non-Hodgkins lymphoma. The wild-type enzymatic activity of the EZH2 complex modifies Lysine 27 with two methyl groups while mutated forms of the EZH2 complex have altered activity. The kit comes in a convenient format, with a 96-well plate for coating with cell lysate, a detection antibody against the dimethylated K27 residue of Histone H3, and HRP labeled substrates. COMPONENTS: Catalog # Component Amount Storage H3 Capture Antibody 25 µg -80 C H3K27me2 Detection antibody 10 µl -80 C 10x Assay buffer 3 ml -80 C Blocking buffer 50 ml -20 C Histone H3K27me2 Control Peptide 20 µg -80 C HRP chemiluminescent substrate A 6 ml +4 C (translucent bottle) HRP chemiluminescent substrate B 6 ml +4 C (brown bottle) 96-well plate 1 plate +4 C (Avoid freeze/ thaw cycles!) MATERIALS OR INSTRUMENTS REQUIRED BUT NOT SUPPLIED: Luminometer or fluorescent microplate reader capable of reading chemiluminescence Rotating or rocker platform RIPA lysis buffer (Pierce, #89900) with protease inhibitor (PI) cocktail (1x) and 1 mm Phenylmethanesulfonyl fluoride (PMSF) BCA Protein Assay Kit (Pierce, #23227) APPLICATIONS: Detection of H3K27(Me2) in cell lysates STABILITY: One year from date of receipt when stored as directed.

2 Example Cell Lysate Protocol: 1) Aspirate Media. Rinse cells 1x with PBS and aspirate. (Keep cells/lysate on ice after rinsing with PBS.) 2) Add 200 μl RIPA lysis buffer directly to well (Pierce #89900 with PI and PMSF) 3) Scrape cells, collect into an Eppendorf tube and sonicate. 4) Precipitate cell debris by centrifugation at 14,000 rpm. for 10 minutes. 5) Move supernatant to a fresh Eppendorf tube, and remove a 5 μl aliquot for protein detection assay. Store remainder of supernatant at -80 o C. 6) Assay the protein concentration of the cell lysate using BCA or Bradford Assay. Note: Lysate will need to be diluted 1:10 in PBS to lower detergent concentration for protein assay to be accurate. ASSAY PROTOCOL: All samples and controls should be tested in duplicate. Step 1: 1) Dilute 1 part 10x Assay Buffer with 9 parts distilled water to make 1x Assay Buffer. Rinse wells with 1x Assay Buffer. 2) Dilute H3 Capture Antibody 1:500 in 1x Assay Buffer. Apply 50 μl diluted H3 Capture Antibody solution to each well. Incubate for 1 hour at 37 o C. 3) Discard supernatant. Wash wells three times with 1x Assay Buffer. After discarding final wash, add 50 μl Blocking Buffer to each well. Incubate overnight at 4 o C. Step 2: 1) Make 2-fold serial dilutions of Histone H3K27me2 Control Peptide in 1x Assay Buffer or lysis buffer. We recommended 50 µl dilutions from 4 µg/ml to µg/ml. 2) Allow microwell strips to equilibrate to room temperature and discard Blocking Buffer.

3 3) Add 50 µl of diluted Histone H3K27me2 Control Peptide to wells designated as Positive Control. 4) Add 50 μl of undiluted or diluted cell lysate to each well designated as Experimental. Note: Cell lysates can be undiluted or serial diluted with 1x Assay Buffer. Include wells with no cells (assay medium only) as a Blank control. Seal wells with tape, and then incubate plate for 2 hours at 37 o C. 5) Wash plate three times with 100 μl 1x Assay Buffer and incubate in 50 μl Blocking Buffer for 10 min. 6) Dilute Detection Antibody 1:1000 in Blocking Buffer. Add 50 μl of diluted Detection Antibody solution to each well. Seal wells with tape and incubate for 1 hour at 37 o C. 7) Wash plate three times with 100 μl 1x Assay Buffer and incubate in 50 μl Blocking Buffer for 10 min. 8) Just before use, mix on ice 50 µl HRP chemiluminescent substrate A and 50 µl HRP chemiluminescent substrate B and add 100 µl per well. Discard any unused chemiluminescent reagent after use. 9) Immediately read sample in a luminometer or microtiter-plate capable of reading chemiluminescence. Blank value is subtracted from all readings. Reading Chemiluminescence: Chemiluminescence is the emission of light (luminescence) which results from a chemical reaction. The detection of chemiluminescence requires no wavelength selection because the method used is emission photometry and is not emission spectrophotometry. To properly read chemiluminescence, make sure the plate reader is set for LUMINESCENCE mode. Typical integration time is 1 second, delay after plate movement is 100 msec. Do not use a filter when measuring light emission. Typical settings for the Synergy 2 BioTek plate reader are: use the hole position on the filter wheel; Optics position: Top; Read type: endpoint. Sensitivity may be adjusted based on the luminescence of a control assay without enzyme (typically we set this value as 100).

4 Example of Assay Results: Detection of Histone H3K27me2 Control Peptide Chemiluminescence Peptide µg/ml Coat Detection of Histone H3K27me2 Control Peptide using the BPS Histone H3 Dimethyl-K27 Detection Kit, cat. # Data shown is lot-specific. For information on specific lots, please contact BPS Bioscience, Inc. at info@bpsbioscience.com

5 RELATED PRODUCTS Product Name Catalog # Size EZH1/EED/SUZ µg EZH1/EED/SUZ12/RbAp48/AEBP µg EZH2/EED/SUZ µg EZH2/EED/SUZ12/RbAp48/AEBP µg EZH2 (R679C)/EED/SUZH12/RbAp48/AEBP µg EZH2 (E740K)/EED/SUZH12/RbAp48/AEBP µg EZH2 (F667I)/EED/SUZH12/RbAp48/AEBP µg EZH2 (A677G)/EED/SUZH12/RbAp48/AEBP µg EZH2 (A677T)/EED/SUZH12/RbAp48/AEBP µg EZH2 (A687V)/EED/SUZH12/RbAp48/AEBP µg EZH2 (del153y)/eed/suzh12/rbap48/aebp µg EZH2 (H689Y)/EED/SUZH12/RbAp48/AEBP µg EZH2 (P132S)/EED/SUZH12/RbAp48/AEBP µg EZH2 (Y641H)/EED/SUZH12/RbAp48/AEBP µg EZH2 (Y641C)/EED/SUZH12/RbAp48/AEBP µg EZH2 (Y641F)/EED/SUZH12/RbAp48/AEBP µg EZH2 (Y641N)/EED/SUZH12/RbAp48/AEBP µg EZH2 (Y641S)/EED/SUZH12/RbAp48/AEBP µg EZH2 Chemiluminescent Assay Kit 52009L 96 reactions EZH2 (Y641F) Chemiluminescent Assay Kit reactions EZH2 (Y641N) Chemiluminescent Assay Kit reactions EZH2 (A677G) Chemiluminescent Assay Kit reactions EZH2 Homogeneous Assay Kit reactions EZH2 (Y641N) TR-FRET Assay Kit reactions Histone H3 K27cMe µg Histone H3 K27cMe µg Histone H3 K27cMe µg Histone H3 K27C µg Anti H3K27me1 polyclonal µg Anti H3K27me2 polyclonal µg Anti H3K27me2/3 monoclonal µg Anti H3K27me3 polyclonal µg Anti H3K27me3S28p polyclonal µg

6 TROUBLESHOOTING GUIDE Problem Possible Cause Solution Luminescence signal of positive control reaction is weak Luminescent signal is erratic or varies widely among wells Background (signal to noise ratio) is high Antibody reaction is insufficient Incorrect settings on instruments Chemiluminescent reagents mixed too soon Inaccurate pipetting/technique Increase time for primary antibody incubation. Avoid freeze/thaw cycles of antibodies. Refer to instrument instructions for settings to increase sensitivity of light detection. Chemiluminescent solution should be used within 15 minutes of mixing. Ensure both reagents are properly mixed. Run duplicates of all reactions. Use a multichannel pipettor. Use master mixes to minimize errors. Bubbles in wells Pipette slowly to avoid bubble formation. Tap plate lightly to disperse bubbles; be careful not to splash between wells. Insufficient washes Increase number of washes. Increase wash volume. Increase Tween-20 concentration to 0.1% in TBST. Results are outside the linear range of the assay Use different concentrations of cell lysate to create a standard curve.