Array Analyze Software
Format: PC compatible
Applications: For use with the MODified™ Histone Peptide Array (Catalog Nos. 13001 & 13005)
Description: The Array Analyze software program is designed for use with the MODified™ Histone
Peptide Arrays (Cat. Nos. 13001 & 13005). The software program will analyze the intensity of spot
interactions from the MODified Histone peptide array and generate a graphical analysis of the histone
peptide modification interactions. The Excel file containing the list of histone modifications and theirassociated 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. For added
convenience, up to three individual histone modifications can be displayed in superposition to theexperimental data.
Installation: The software is designed for use on PCs only. Download the software file to yourcomputer desktop, and unzip the file. Open the unzipped folder and begin installation by running thesetup.exe file. If you get an error during installation or if the software does not run after installation,
you should first run VisualBasicPowerPacksSetup.exe & MSChart.exe before running setup.exe.
A shortcut for the Array Analyze program should automatically appear on your desktop.
Usage Instructions:
1. Follow the MODified™ Histone Peptide Array manual to perform experiments.
2. Capture an image of the processed array. Crop the image to the size of the slide using normal
exposure settings. Sometimes it helps to over-expose the image to see the edges of the slide.
Make sure to return the exposure settings back to normal before capturing the image.
Note: If there are only a few spots visible, this is a good time to over-expose the slide andidentify them. Even spots that do not react with the antibody should be faintly visible on thearray. Once all the spots are visible, identify the spots that did react and make a note of
their position for use later with the spot assignment in the array software.
3. Save the cropped image as a .tif file.
4. Start the Array Analyze program by clicking the shortcut on your desktop.
5. Select Open Slide…
6. Upload the .tif file.
7. Optionally crop the image if the array spots do not fill the display window.
a. Use the mouse to click-and-drag a rectangle around the border of the array image, making
sure to leave a small amount of space outside of the spots.
b. Click the Crop button. (The original image file will not be modified).

8. Select the option “Dark Spots on Light Background” or “Light Spots on Dark Background” whichcorresponds to your image type.
9. In order to align the array reference grid with the slide you will need to identify two spots. It is best
if spot 1 is in the upper left
corner of the left half of the array and spot 2 is in the lower right corner of the right half of the array.
However, in the case that only a few spots show up, take care to correctly identify their location
and use those spots as the reference spots.
a. Align the cursor with the center of a spot on the upper left side of the left array.
b. Click on the center of this spot.
c. Use the drop down menu to select the coordinates of the selected spot (Row A to P, Column 1to 24).
d. Click the Assign Left Spot button.
e. Repeat for the lower right side of the right array and click the Assign Right Spot button.
10. Once the left and right spots have been assigned, press the Assign Spots button.
11. The grid image will be overlaid onto the array image. Verify the grid is in alignment with the arrayspots. If not, click the Resetbutton and repeat spot alignment. See the example image below.

212. Optionally edit the fields for Chart Subtitles and Experiment Notes. By default these contain theoriginal image name and the date that the original image was saved. These fields will appear asannotations on results graphs.
13. Click the Analyze Array button. A new tabbed Window will open containing the analysis results.
14. To Save any of the results, select one of the Save options from the File menu. Save All saves
results from all tabs, or you may Save the output displayed on each tab individually. By default,
the results will be saved in the same directory as the original image file. The location and namesof the files can be changed if desired by editing the information in the Save dialog. Note that tabs
which have multiple display options will only save the selected option when Save All is used. To
save multiple graphs from a single tab, use the Save As option under the File menu.
15. The Spot Alignment tab contains an image of the grid overlay. If saved, this image will be called“name_slide.png”.
16. The Spot Statistics tab gives a comparison of the intensities of each spot in the left and right array(left image). The data points ideally should be on a straight line connecting 0,0 and 1,1 whichindicates perfect duplicates. Outliers (or poor replicates) will not fall along the linear line. If saved,
this figure will be called “name_figXY.png”. The distribution errors (right image) displays the errorsof the intensities between the right and left spot, normalized to the maximum intensity. It is ideal tohave most of the peptides contain an error range of 0-5% (which means very little variation
between the left and right sides). If saved, this figure will be called “name_fig1.png”.
17. The Background Setting tab allows you to define the background for the calculation of theSpecificity, Reactivity, and Modification Analysis profiles. Click on the graph to manually change
the background level (orange line on graph), or select the Use Automatic Background Level button
for the default background values. Lowering the background level will decrease the specificity of
the analysis allowing weakly associated spots to be included, while raising the background level
will increase the specificity of the analysis so only spots with higher signal levels will be3

considered positive. If saved, this figure will be called “name_fig2.png”.
18. The Specificity tab displays a background subtracted graph of the 10 modifications with thegreatest specificity factors. While all modifications have been accounted for in determining thespecificity factor, only the 10 modifications with the highest specificity factor values are graphed.
The larger the difference in specificity factor values for the mark of interest versus other marks,
the more specific the interaction. The Multiple Peptide Average option calculates the ratio of theaverage intensity of all spots containing the mark divided by the average intensity of all spots notcontaining the mark. Non-specific signals will decrease the specificity factor values. These nonspecific signals can be caused by improper antibody dilutions, over development, and non-specificbinding (false positives). Note that the data used to generate this graph can be viewed and savedin the Raw Data tab (see below). The Single Peptide Intensity option displays only single modified
peptides without averaging all spots containing the mark. If saved, the graph will be called“name_fig3.png”. See the example graphs below.
Note:
When using the Multiple Peptide Average view to interpret the specificity of your binding
event, it is important to understand that this information is provided as a general overview ofspecificity, but the graph should not be considered all inclusive of the information needed toassess complete specificity. The presence of "false positives" and "false negatives" will alter theaverage intensity values and can change the specificity factor value for a particular modification.
False positives indicate non-specific binding of a modification that was not anticipated. The
intensity of the false positive spots will be averaged into the denominator of the ratio, resulting in a
reduced specificity factor value. Alternatively, false negatives can result from the inhibition ofbinding due to the presence of a neighboring modification. In this case a peptide that contains themodification of interest will have a lower than expected signal. This reduced intensity value isaveraged into the numerator of the ratio generating a reduced specificity factor value. The result isthat a modification with very specific binding may display a lower specificity factor value than aless specific binding event due to the presence of neighboring modifications.
To address these influences, utilize the Reactivity tab (see below) to look at individualmodifications that show binding. Then use the Modification Analysis tab (see below) and selectthe modifications identified by the Reactivity tab to evaluate the effects of neighboring
modifications on binding specificity.

419. The Reactivity tab displays the background subtracted intensity values for all single modified
peptides, as well as for control spots. Selecting a modification from the pull down menu scales theintensity of the selected modification to 1.0. Select “none” to display unscaled values. The impactof neighboring modifications on the single modified peptides can be further investigated using theModification Analysis tab and selecting for the modification of interest (see below). If saved, thegraph will be called “name_fig6.png”. See the example graph below.

20. The Modification Analysis tab displays background subtracted intensity values for all spots
containing the selected modification. If saved, the graph will be called “name_fig5.png”. See theexample graph below.

521. The Annotated Array tab allows you to select up to three modifications to be displayed insuperposition to the experimental data. Select these modifications from the drop down menus.
Press the Select Most Specific button to automatically display the top 3 modifications from theSpecificity chart. Check the boxes in order to see the array grid overlay or to identify themodifications on both sides of the array. Use the color coding to identify the modifications. If saved,
this graph will be called “name_fig4.png”. See the example chart below.

22. The Raw Data tab displays the comma separated value (CSV) data for the peptide intensities forall spots, the ranked intensities for all spots, and the ranked modifications. This CSV data can besaved in Excel compatible files to allow for individual analysis. If saved, these files will be called“name_data.csv”, “name_ranked_list.csv”, and “name_ranked_modifications.csv”, respectively.
The data in the “name_data.csv” file is the raw data for the array. The fields are:a. Peptide No.

the peptide number (1 through 384)

b. Location

the grid coordinates of the spot on the array (A1 through P24)

c. Namethe peptide name6

d. Mod 1

the first modification, if present

e. Mod 2

the second modification, if present

f. Mod 3

the third modification, if present

g. Mod 4

the fourth modification, if present

h. Intensity lefti.

Intensity rightj.

Intensity av.

the intensity value for the spot on the left side of the arraythe intensity value for the spot on the right side of the array
the average between the duplicate (left and right) spotsk. Error
the error between the duplicates (the lower the value, the better theduplicate readings. This isvisually depicted as figXY)
The data in the “name_ranked_list.csv” file is ranked according to intensity values from highest tolowest. The control and background spots on the array should be found ranked towards thebottom if there was low background and good specificity. The fields are:a. Rank

the peptide rank (1 through 384)

b. Pept. No.

the peptide number (1 through 384)

c. activity

the average intensity value from the name_data.csv filed. norm. act.
the normalized activity. This is calculated by dividing the activity by theactivity of the number one
ranked peptide.
e. The remainder of the fields is the data for each modification, with a value of 1 indicating themodification is present in the peptide, and a value of zero indicating that the modification
was not present.
The data in the “name_ranked_modifications.csv” file list each modification ranked by specificity
factor, from highest to lowest. This is the same data used to generate the Multiple Peptide
Average chart. The fields are:a. Rankb. Modification

the rank of the modification
the name of the modificationc. No pos

number of peptides on the array that contain the modificationd. No neg

number of peptides on the array that do not contain the modificatione. Av Int pos

average intensity of all spots containing the modification

f. Av Int neg
g. Specaverage intensity of all spots that do not contain the modification
Specificity factor (Av Int Pos divided by Av Int Neg)

23. The Protocol Log tab displays a log of steps used to perform the analysis. If saved, this log will becalled “name_protocol.txt”.
24. To start a new analysis or edit the analysis of the current image, select Open Image… from theFile menu.
25. Close out of the program by selecting Exit from the File menu, or by using the X in the upper rightcorner of the program.

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