Human/Mouse Acetylated-Histone H3 and Histone H3 ELISA

RayBio® Human/Mouse Acetylated-Histone H3 ELISA Kit. This ELISA is for measuring Acetylated-Histone H3, as well as Histone-H3 in human and mouse cell lysates.

PTE-ACEHIS3-T-1
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Antigen Information

Accession Number:
  • P84243
  •  P84244
Gene ID:
  • 3020
  • 3021
Gene Symbols:
  • H3.3A
  • H3.3B
  • H3F3
  • H3F3A
  • H3F3B
  • PP781
Protein Name & Synonyms:
Histone H3.3
Target Species:
  • Human
  • Mouse

Assay Format

Specificity:
The antibody pair provided in this kit recognizes human/mouse Acetylated-Histone H3, as well as Histone-H3.
Number of Targets Detected:
2
Species Detected:
  • Human
  • Mouse
Compatible Sample Types:
  • Cell Lysates
  • Tissue Lysates
Design Principle:
  • Sandwich-based
Method of Detection:
Colorimetric
Quantitative/Semi-Quantitative:
Semi-Quantitative
Solid Support:
96-well Microplate

Product Specifications

Size:
1, 2, or 5 x 96-Well Strip Microplate Kit

Introduction

RayBio® Acetylated-Histone H3 and Histone H3 ELISA kit is a very rapid, convenient and sensitive assay kit that can monitor the activation or function of important biological pathways in human cell lysates. By determining Acetylated Histone H3 protein in your experimental model system, you can verify pathway activation in your cell lysates. You can simultaneously measure numerous different cell lysates without spending excess time and effort in performing a Western Blotting analysis.

This Sandwich ELISA kit is an in vitro enzyme-linked immunosorbent assay for the measurement of human Acetylated-Histone H3 and Histone H3. An anti-pan Histone H3 antibody has been coated onto a 96-well plate. Samples are pipetted into the wells and Histone H3 present in a sample is bound to the wells by the immobilized antibody. The wells are washed and rabbit anti-Acetylated-Histone H3 antibody is used to detect Acetylated Histone H3 or mouse anti-Histone H3 antibody is used to detect Histone H3. After washing away unbound antibody, HRP-conjugated anti-rabbit IgG or HRP-conjugated anti-mouse IgG is pipetted to the wells. The wells are again washed, a TMB substrate solution is added to the wells and color develops in proportion to the amount of Acetylated Histone H3 or Histone H3 bound. The Stop Solution changes the color from blue to yellow, and the intensity of the color is measured at 450 nm.

Product Features

  • Simultaneously measure acetylated protein and pan protein in one experiment (for normalization purpose)
  • Screen numerous different cell lysates without performing a Western Blot analysis
  • Minimal hands-on time, convenient, and non-radioactive material

Application Notes

Kit Components
  • Pre-Coated 96-well Strip Microplate
  • Wash Buffer
  • Anti-acetylated Antibody
  • Anti-Pan Antibody
  • HRP-Conjugated Secondary Antibody
  • Streptavidin-Conjugated HRP
  • Assay Diluent
  • TMB One-Step Substrate
  • Stop Solution
  • Lysis Buffer
  • Positive Control Sample
Other Materials Required
  • Distilled or deionized water
  • 100 ml and 1 liter graduated cylinders
  • Tubes to prepare sample dilutions
  • Protease and Phosphatase inhibitors
  • Precision pipettes to deliver 2 µl to 1 ml volumes
  • Adjustable 1-25 ml pipettes for reagent preparation
  • Benchtop rocker or shaker
  • Microplate reader capable of measuring absorbance at 450 nm
Protocol Outline
  1. Prepare all reagents and samples as instructed in the manual.
  2. Add 100 µl of sample or positive control to each well.
  3. Incubate 2.5 h at RT or O/N at 4 °C.
  4. Add 100 µl of prepared primary antibody to each well.
  5. Incubate 1 h at RT.
  6. Add 100 µl of prepared 1X HRP-Streptavidin to each well.
  7. Incubate 1 h at RT.
  8. Add 100 µl of TMB One-Step Substrate Reagent to each well.
  9. Incubate 30 min at RT.
  10. Add 50 µl of Stop Solution to each well.
  11. Read at 450 nm immediately.

Typical Data

Positive Control
COS cells were treated with Trichostatin A (TSA). Solubilize cells at 4 x 107 cells/ml in Cell Lysate Buffer. Serial dilutions of lysates were analyzed in this ELISA. Please see step 3 of Part VI Reagent Preparation for detail.
TSA Stimulation of COS Cell Line
COS cells were treated or untreated with TSA. Cell lysates were analyzed using this ELISA and Western Blot.


Storage/Stability

Upon receipt, the kit should be stored at –20°C. Please use within 6 months from the date of shipment. After initial use, Wash Buffer Concentrate (Item B), Assay Diluent (Item E), TMB One-Step Substrate Reagent (Item H), Stop Solution (Item I) and Cell Lysate Buffer (Item J) should be stored at 4°C to avoid repeated freeze-thaw cycles. Return unused wells to the pouch containing desiccant pack, reseal along entire edge, and store at –20°C. Item D, store at 2-8°C for up to one month (store at -20°C for up to 6 months, avoid repeated freeze-thaw cycles). Reconstituted Positive Control (Item K) should be stored at -70°C.

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Usage

This product is furnished for LABORATORY RESEARCH USE ONLY.
Not for diagnostic or therapeutic use.

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