MODified™ Histone Peptide Array Data

Select antibody images and graphs are available below. To see a complete list of all the Active Motif antibodies that have been tested using the MODified™ Histone Peptide Array, please follow the link to array tested.

 

Figure 1: Image of ECL detection of Histone H3 trimethyl Lys9 (H3K9me3) pAb.

Active Motif's Histone H3 trimethyl Lys9 (H3K9me3) pAb (Catalog No. 39161) was used at 1:2,000 dilution on the MODified Histone Peptide Array. Anti-rabbit HRP secondary antibody was used at 1:2500 dilution, followed by ECL detection and image capture with a CCD camera.

 

Figure 2: Graphical analysis of Histone H3 trimethyl Lys9 (H3K9me3) pAb.

Spot densitometry was used to analyze spot intensity from the ECL camera image. Results were compared to the provided reference grid to determine the associated peptide content. The results were graphed as a specificity factor, which is the ratio of the average intensity of all spots containing the mark divided by the ratio of the average intensity of all spots not containing the mark. The results show Active Motif's Histone H3 trimethyl Lys9 pAb (Catalog No. 39161) has very little cross-reactivity with other histone modifications.

 

Figure 3: Image of ECL detection of Histone H3 dimethyl Lys4 (H3K4me2) pAb.

Active Motif's Histone H3 dimethyl Lys4 (H3K4me2) pAb (Catalog No. 39141) was used at 1:10,000 dilution on the MODified Histone Peptide Array. Anti-rabbit HRP secondary antibody was used at 1:2500 dilution, followed by ECL detection and image capture with a CCD camera.

 

Figure 4: Graphical analysis of Histone H3 dimethyl Lys4 (H3K4me2) pAb.

Spot densitometry was used to analyze spot intensity from the ECL camera image. Results were compared to the provided reference grid to determine the associated peptide content. The results were graphed as a specificity factor, which is the ratio of the average intensity of all spots containing the mark divided by the ratio of the average intensity of all spots not containing the mark. The results show Active Motif's Histone H3 dimethyl Lys4 pAb (Catalog No. 39141) has very little cross-reactivity with other histone modifications.

 

Figure 5: Graphical analysis of Histone H4 acetyl Lys16 (H4K16ac) pAb.

Spot densitometry was used to analyze spot intensity from the ECL camera image. Results were compared to the provided reference grid to determine the associated peptide content. The results were graphed as a specificity factor, which is the ratio of the average intensity of all spots containing the mark divided by the ratio of the average intensity of all spots not containing the mark. The results show Active Motif's Histone H4 acetyl Lys16 pAb (Catalog No. 39167) has very little cross-reactivity with other histone modifications.

MODified™ Histone Peptide Array Advantages

• Histone specific - unique array panel specific for histones and histone modifications
• Multiple combinantions - peptides contain up to four modification combinations each
• Detects like a Western blot - works with either ECL-based or colorimetric detection systems
• Versatility - use the arrays to study antibody, protein or enzyme interactions

How do the MODified™ Histone Peptide Arrays Work?

To generate MODified Arrays, Active Motif synthesizes a series of 19mer histone H2A, H2B, H3 and H4 peptides, each of which may contain as many as four modifications per peptide, on a modified cellulose support which can be dissolved after synthesis. The solutions of peptides covalently linked to macromolecular cellulose are then spotted in duplicate onto a glass slide as a three-dimensional layer. This high peptide density in the spots is advantageous for identifying protein-interaction sites with low binding constants. The peptide coated slides contain 768 spots (384 modification combinations in duplicate) and are offered as single arrays (Catalog No. 13001) or as a pack of five arrays (Catalog No. 13005).

The MODified Histone Peptide Arrays can be used to screen antibodies, enzymes and proteins for cross reactivity or binding interactions with histones and their post-translational modifications. Following a blocking step, the array is incubated with either antibodies, proteins or enzymes for one hour, washed and then incubated for one hour with either a horseradish peroxidase (HRP) or alkaline phosphatase (AP) conjugated secondary antibody. Developing solution is then added and the image is captured using film or a CCD camera; no special equipment is needed.

Figure 1: Flow chart of the MODified Histone Peptide Array.

This flow chart illustrates the use of the MODified Histone Peptide Arrays (Click image to enlarge.)

MODified Histone Peptide Array Analysis

Active Motif's FREE Array Analyze Software can be used to analyze the spot intensities on the array and generate a graphical analysis of the histone peptide modification interactions. The Excel file containing the list of histone modifications and their associated locations has been incorporated into the software to help automate the identification process for the arrays. With the Array Analyze Software, information about spot intensity, averages and errors can be saved in Excel-compatible files to allow for individual analysis. Additionally, up to three individual histone modifications can be displayed in superposition to the experimental data, enabling better visualization of the effects of neighboring modifications. Click here to download the Array Analyze Software Manual and the FREE Array Analyze Software.

MODified Histone Peptide Array Specifications:

• Standard microscope slides (26x76 mm, white coating)
• 768 spots per slide (384 peptide-conjugate spots printed in duplicate)
• Spot-to-spot distance 1.2 mm
• Peptides are covalently bound to cellulose via C-terminus
• Arrays contain control peptides and location marks

To download a list of histone modifications offered on the MODified Histone Peptide array and the corresponding peptide location on the array, CLICK HERE.

Contents & Storage

The MODified Histone Peptide Arrays consist of either a single array slide or a five pack of slides. CLICK HERE to download the reference Excel file containing the grid pattern and associated peptide content. A protocol for antibody screening using either ECL or colorimetric detection is also included, or can be downloaded under the Documents tab.

The MODified Histone Peptide Arrays can be stored at 4°C for up to three months. For long-term storage of up to six months, we recommend keeping the arrays at -20°C.

The MODified Array Labeling kit contains Blocking Buffer AM2, 10X Wash Buffer AM4, ECL Reagent A, ECL Reagent B as well as c-Myc mouse monoclonal antibody to detect the positive control c-Myc peptide on the array and anti-rabbit and anti-mouse HRP-conjugated secondary antibodies. The MODified Histone Peptide Arrays are NOT included in the kit. Reagent storage conditions vary from 4°C to -20°C, see manual for details. All reagents are guaranteed stable for 6 months when stored properly.