RayBio Antibody Array Analysis Tool

RayBio

Antibody Array Analysis Tool

©2004 RayBiotech, Inc. All rights reserved.

RayBio® is registered trademark of RayBiotech, Inc. Unauthorized copying, reproduction prohibited.

Overview of RayBio® Antibody Array Analysis Tool.

This Analysis Tool is a data analysis program specifically designed for analysis of RayBio® Antibody Arrays. This tool will assist you in the following aspects:

• locate your signal intensities to antibody array map

• direct link to a website containing more detailed information on the particular cytokine

• protein list sorting

• average signal intensities

• subtract background

• normalize the data from different samples

• obtain protein level comparison charts among different samples

This program is operated based on MS Excel technology. It is user friendly and requires no professional training.

Process Flow

Array membrane

Experimental Procedure

Visualization of signal

Measurement of signal intensity

Importing data to RayBio®_{Antibody array Analysis tool}

Aligning data into RayBio®_{Antibody array map}

Obtain result 20 18 3 8 19 6 19 3 2 1 3 4 20 18 3 8 19 6 19 3 2 1 3 4

To use RayBio® antibody array analysis tool, you must measure the signal intensities of the array membranes by using an imaging system. Two ways can achieve the desired results.

1. If the signals in the array membranes are imaged and quantified by chemiluminiscence imaging system, the raw data can be copied and pasted to RayBio® antibody array analysis tool.

2. If the signals in the array membranes are captured by x-ray film, use a densitometer to quantify the signal intensities, followed by copying and pasting the raw data to RayBio® antibody array analysis tool.

Data analysis step by step

1. Launch RayBio

antibody array Analysis tool

application

(Microsoft excel-based

program).

a. Imaging the signal in the array membrane with corresponding program such as chemiluminescence imaging instrument (UVP, Alpha-innotech, Kodak, Fuji, Bio-Rad and so on) or densitometry scan. Retrieve signal intensities by importing the data.

b. Click on importing data on the bottom worksheet.

c. Copy the raw data from the corresponding imaging program (for example the data from UVP chemiluminescence instrument), and paste to importing data page.

3. Aligning data

Since different imaging programs have different formats, it is important to correctly align the data into our map. After this step has been completed, RayBio® antibody array analysis tool will do all analysis for you.

Copy the data from importing data:

Click on aligning data on the bottom worksheet. Paste the data to the designated area.

Continue to copy and paste all data to designated area.

For background, select several blanks (do not select the blanks next to the positive), copy and paste the background from “importing data” to “aligning data”.

4. Map and antibody list

C list on the bottom worksheet, the data you generate from the imaging instruction

. Sorting

will appear on our array map. Now, you can find the signal intensity corresponding to the protein of interest.

Click on sorting on the bottom worksheet. he data will be arranged in alphabetic order.

. Average

Click on average on the bottom worksheet, the averaged data from two spots will appear.

. Background subtraction

9

Note: The background from Aligning data is averaged. The signal intensities for every protein are subtracted from the averaged background.

10. Normalization 1: Normalize background subtraction data

The data after background subtraction will be normalized according to positiv ontrol of the first sample will be considered as 1 and the signal intensities of

e control densities. The positive other samples will be calculated f particular spot= signal intensity of particular spot x

1/ sample in particular spot

1. Chart 1: from Normalization 1

according to the formula: normalized signal intensity o ostive positve signal intensity ). (p

Click on Chart 1 on the bottom worksheet. The signal intensities of particular proteins from different samples data 1.

2.

Normalization 2: Normalize data without subtraction background

will be compared according to normalization

1

The data without subtraction background will be normalized according to positive control densities. The ositive control of first sample will be considered as 1 and the signal intensities of other samples will be

tensity of particular spot= signal intensity of particular

1/ sample in particular spot).

13. Chart 2: from Normalization 2

calculated according to formular: normalized signal in ot x (postive positve signal intensity

sp

This function provides an option for investigators who prefer to normalize the data without background subtraction

Click on Chart 2 on the bottom worksheet. The signal intensities of particular proteins from different samples ill be compared according to normalization data 2.

otice:

Finally, save the analyzed data on your computer.

1. This analysis tool will greatly reduce your time and effort in analyzing data. However, this program is neither bioinformatics software, nor a statistics tool. You may still need bioinformatics and statistics to further

nalyze the data.

value is a challenging problem in analyzing array data, particularly the low intensity signals. Since ifferent investigators apply different instruments to measure the signal intensity, it is hard for us to provide

. RayBio is registered trademark of RayBiotech, Inc. Microsoft Excel is registered trademark of Microsoft

mitted in any form without the written ermission of RayBiotech, Inc.

a a

2. Cut-off d

exact cut-off value for the arrays. As a rule of thumb, if you can see the signal by your eye, it is a real signal. Otherwise, you may apply bioinformatics to determine the cut-off value.

®

3

Corporations.

4. All right are reserved for both RayBio® antibody analysis tool and instruction. No part of this RayBio® antibody array analysis tool or its instructions may be reproduced or trans