ab Histone H2B Acetyl (K5) ELISA Kit

Transcription

1 ab Histone H2B Acetyl (K5) ELISA Kit Instructions for Use For the quantitative measurement of acetylated lysine (K5) on Histone H2B in cell lysates. This product is for research use only and is not intended for diagnostic use. Version 1 Last Updated 4 December 2014

2 Table of Contents INTRODUCTION 1. BACKGROUND ASSAY SUMMARY GENERAL INFORMATION 3. PRECAUTIONS 3 4. STORAGE AND STABILITY 4 5. MATERIALS SUPPLIED 4 6. MATERIALS REQUIRED, NOT SUPPLIED 5 7. LIMITATIONS 5 8. TECHNICAL HINTS 6 ASSAY PREPARATION 9. REAGENT PREPARATION PLATE PREPARATION ASSAY PROCEDURE 11. ASSAY PROCEDURE 9 DATA ANALYSIS 12. TYPICAL DATA 10 RESOURCES 13. TROUBLESHOOTING NOTES 1

3 INTRODUCTION 1. BACKGROUND Abcam s Histone H2B Acetyl (K5) RabMab in vitro ELISA (EnzymeLinked Immunosorbent Assay) kit is designed for accurate quantitative measurement of acetylated lysine (K5) on Histone H2B in cell lysates. Abcam s Histone H2B Acetyl (K5) RabMab ELISA Kit employs a sandwich enzyme immunoassay technique to detect endogenous levels of Histone H2B when acetylated at Lys5. Rabbit anti-histone H2B monoclonal RabMab antibody has been pre-coated onto a microplate. Cell lysate is pipetted into the wells. Following extensive washing, biotinylated rabbit anti-acetyl-histone H2B (K5) monoclonal RabMab antibody reagent is added. Following a wash to remove any unbound antibody reagent, streptavidin-hrp is added to the well. After washing away the unbound streptavidin-hrp, a substrate solution is added to the wells to develop color. The magnitude of the absorbance for this developed color is proportional to the amount of acetyl-histone H2B (K5). Changes in chromatin structure play a key role in the regulation of transcription in eukaryotes. The nucleosome is the primary building block of chromatin, and is made up of four core histone proteins (H2A, H2B, H3 and H4). Histones function as a structural component in the nucleus and as a regulator of the gene expression. Acetylation of core histones regulates gene expression. Histone H2B is acetylated in the N-terminal region, primary at lysines 5, 12, 15, and 20. Ubiquitylation of histone H2B at lysine 123 by Rad6 has been implicated in transcriptional activation and lysine methylation on histone H3. Histone H2B phosphorylation at Ser10 (for yeast) and Ser14 (for mammalian cells) has been linked to regulation of cell apoptosis and meiosis. Histone modifications promote or prevent binding of proteins that drive particular region of the genome into active transcription or repression. 2

4 INTRODUCTION 2. ASSAY SUMMARY Equilibrate all reagents to room temperature. Prepare all the reagents, samples, and standards as instructed. Add standard or sample to each well used. Add biotinylated labeled detector RabMab antibody to each well used. Incubate at room temperature. Aspirate and wash each well. Add prepared Streptavidin-HRP mix to each well. Incubate at room temperature. Aspirate and wash each well. Add the TMB Solution to each well until color develops and then add the Stop Solution. Immediately begin recording the color development. 3

5 GENERAL INFORMATION 3. PRECAUTIONS Please read these instructions carefully prior to beginning the assay. All kit components have been formulated and quality control tested to function successfully as a kit. Modifications to the kit components or procedures may result in loss of performance. 4. STORAGE AND STABILITY Store kit at +2-8ºC immediately upon receipt. Refer to list of materials supplied for storage conditions of individual components. Observe the storage conditions for individual prepared components in the Reagent Preparation section. 5. MATERIALS SUPPLIED Item Amount Anti-Histone H2B Ab Coated Microplate Biotinylated Anti-Acetyl Histone H2B (K5) Antibody Reagent (1X) Streptavidin-HRP Reagent (100X) 1 x 96 well plate Storage Condition (Before Preparation) +2-8 C 1 x 11 ml C 1 x 120 µl +2-8 C Antigen/Antibody Diluent Buffer (1X) 1 x 20 ml C ELISA Washing Buffer (10X) 1 x 25 ml +2-8 C TMB A Substrate Solution (1X) 1 x 7 ml C TMB B Substrate Solution (1X) 1 x 7 ml +2-8 C Stop Solution (1X) 1 x 11 ml C 4

6 GENERAL INFORMATION 6. MATERIALS REQUIRED, NOT SUPPLIED These materials are not included in the kit, but will be required to successfully utilize this assay: Deionized water Vortex mixer or equivalent Pipettors and pipette tips of various sizes Rotating shaker Microtiter plate reader 7. LIMITATIONS Assay kit intended for research use only. Not for use in diagnostic procedures. Do not use kit or components if it has exceeded the expiration date on the kit labels. Do not mix or substitute reagents or materials from other kit lots or vendors. Kits are QC tested as a set of components and performance cannot be guaranteed if utilized separately or substituted. 5

7 GENERAL INFORMATION 8. TECHNICAL HINTS Samples generating values higher than the highest standard should be further diluted in the appropriate sample dilution buffers. Avoid foaming components. Avoid cross contamination of samples or reagents by changing tips between sample, standard and reagent additions. Ensure plates are properly sealed or covered during incubation steps. Complete removal of all solutions and buffers during wash steps. This kit is sold based on number of tests. A test simply refers to a single assay well. The number of wells that contain sample, control or standard will vary by product. Review the protocol completely to confirm this kit meets your requirements. Please contact our Technical Support staff with any questions. or bubbles when mixing or reconstituting 6

8 ASSAY PREPARATION 9. REAGENT PREPARATION Equilibrate all reagents and samples to room temperature (1825 C) prior to use. Store all buffers and reagents at 4 C when not in use X Streptavidin-HRP Reagent Equilibrate Streptavidin-HRP Reagent (100X) to room temperature before diluting to 1X with deionized water X ELISA Washing Buffer Equilibrate ELISA washing buffer (10X) to room temperature before diluting to 1X with deionized water X Biotinylated Anti-Acetyl Histone H2B (K5) Dilute 50X Biotinylated Anti-Acetyl Histone H2B (K5) Antibody with Antigen/Antibody Diluent Buffer (1X). 7

9 ASSAY PREPARATION 10. PLATE PREPARATION The 96 well plate strips included with this kit are supplied ready to use. It is not necessary to rinse the plate prior to adding reagents. For each assay performed, a minimum of 2 wells must be used as blanks, omitting primary antibody from well additions. For statistical reasons, we recommend each sample should be assayed with a minimum of two replicates (duplicates). Well effects have not been observed with this assay. Bring stripped microtiter plate to room temperature. Keep appropriate numbers of strips for an experiment and remove extra strips from microtiter plate by evenly pushing the bottoms of the microwell strips. Replace unrequired strips immediately into the bag, seal and store at 4 C. 8

10 ASSAY PROCEDURE 11. ASSAY PROCEDURE Equilibrate all materials and prepared reagents to room temperature prior to use. It is recommended to assay all standards, controls and samples in duplicate Prepare all reagents, working standards and samples as directed in the previous section Remove excess microplate strips from plate and return to foil pouch Add 100 µl of each diluted cell lysate to the appropriate well. Incubate for 1 hr. at room temperature on a shaker Aspirate each well and wash 3 times with 200 µl 1X Wash Buffer per well Add 100 µl of Biotinylated Anti-Acetyl-Histone H2B (K5) Antibody Reagent (1X) to each well. Incubate for 1 hour at room temperature on a shaker Aspirate each well and wash with 200 µl 1X Wash Buffer per well. Repeat wash 2 additional times Add 100 µl of Streptavidin-HRP Reagent to each well. Incubate for 30 minutes at room temperature on a shaker Aspirate each well and wash with 200 µl 1X Wash Buffer per well. Repeat wash 2 additional times Combine TMB A and TMB B (1:1). Add 100 µl of combined substrate solution to each well. Incubate for 30 minutes (max) at room temperature on a shaker. Note: Volume of each 50μl x (number of wells +1) TMB substrate needed = Add 100 µl of Stop Solution to each well Determine the optical density at 450 nm using a microplate reader within 30 minutes. 9

11 DATA ANALYSIS 12. TYPICAL DATA ab Histone H2B Acetyl (K5) ELISA Kit detects levels of acetylated lysine (K5) on Histone H2B. When A431 cells were treated with TSA, the levels of total Histone H2B protein remain unchanged, as shown by Western blot analysis (Figure 1). However, TSA increases levels of acetylated H2B, as shown by Western blot analysis (Figure 1) and sandwich ELISA (Figure 2). TSA + - TSA + - Figure 1. Treatment of A431 cells with TSA (500 ng/ml for 4 hr) increases the acetylation of Histone H2B at Lys5. Figure 2. The relationship between the protein concentration of lysates from untreated and TSA-treated A431 cells and assay optical density readings. 10

12 RESOURCES 13. TROUBLESHOOTING Problem Cause Solution Poor standard curve Improper standard dilution Confirm dilutions made correctly Standard improperly reconstituted (if applicable) Briefly spin vial before opening; thoroughly resuspend powder (if applicable) Standard degraded Store sample as recommended Curve doesn't fit scale Try plotting using different scale Incubation time too short Try overnight incubation at 4 C Target present below detection limits of assay Decrease dilution factor; concentrate samples Precipitate can form in wells upon substrate addition when concentration of target is too high Increase dilution factor of sample Using incompatible sample type (e.g. serum vs. cell extract) Detection may be reduced or absent in untested sample types Sample prepared incorrectly Ensure proper sample preparation/dilution Wells are insufficiently washed Wash wells as per protocol recommendations Contaminated wash buffer Make fresh wash buffer Low signal High background 11

13 RESOURCES Problem Large CV Low sensitivity Cause Solution Waiting too long to read plate after adding STOP solution Read plate immediately after adding STOP solution Bubbles in wells Ensure no bubbles present prior to reading plate All wells not washed equally/thoroughly Check that all ports of plate washer are unobstructed/wash wells as recommended Incomplete reagent mixing Ensure all reagents/master mixes are mixed thoroughly Inconsistent pipetting Use calibrated pipettes and ensure accurate pipetting Inconsistent sample preparation or storage Ensure consistent sample preparation and optimal sample storage conditions (e.g. minimize freeze/thaws cycles) Improper storage of ELISA kit Store all reagents as recommended. Please note all reagents may not have identical storage requirements. Using incompatible sample type (e.g. Serum vs. cell extract) Detection may be reduced or absent in untested sample types 12

14 RESOURCES 14. NOTES 13

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