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2D DIGE Cross-Gel Data Analysis

2D DIGE In-Gel Data Analysis

 

 

 

 

The 2D DIGE Cross-Gel Data Analysis is ideal for comparing multiple 2D DIGE gels, with more than 3 samples, or multiple replicates in each experimental group.

An 'Internal Standard' containing equal amount of protein of each sample is run on all gels.

Importance of Internal Standard
o Represents all protein spots
o Decreases gel-to-gel variation and enables gel-to-gel matching
o Enables fully automated, accurate statistical analysis of each spot
o Allows detection of consistent biological variance

How many replicates do I need?
o Cell lines: 4
o Mice: 4+
o Humans: more are better within the constraints of reality

Study Designs (Click to see more details)
o Study Design 1: More than 3 individual samples
o Study Design 2: Two groups of samples, multiple replicates per group
o Study Design 3: Two groups of samples, Two conditions per group, multiple replicates
o Study Design 4: Multiple groups of samples, multiple conditions per group, multiple replicates

Study Design 1: Example

 

Type of samples
o Four individual samples: sample 1, 2, 3, 4
o No replicates

Number of gels: 2

Data analysis: Pair-wise comparison in any of the 2 samples

Cy2
Cy3
Cy5

Gel 1
Internal Standard
Sample 1 Sample 2
Gel 2
Internal Standard
Sample 3 Sample 4
Internal Standard = Sample 1-4

 

2D DIGE Cross-gel Analysis 2D DIGE Cross-gel Analysis
2D DIGE Cross-gel Analysis

Data Report

Protein expression ratio between any 2 samples

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Study Design 2: Example

 

Type of samples
o Two groups of samples: Control (C) and Test (T)
o Four replicates in each group: C_1, 2, 3, 4; T_1, 2, 3, 4

Number of gels: 4 (Dye swap labeling)

Data analysis: comparison between the two groups

Cy2
Cy3
Cy5

Gel 1
Internal Standard
C_1
T_1
Gel 2
Internal Standard
T_2
C_2
Gel 3
Internal Standard
C_3
T_3
Gel 4
Internal Standard
T_4
C_4
Internal Standard = C1-4, T1-4

 

2D DIGE Cross-gel Analysis 2D DIGE Cross-gel Analysis
2D DIGE Cross-gel Analysis

Data Report

1. Average protein expression ratio between
control and test group

2. Statistical analysis of Control vs. Test

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Study Design 3: Example

 

Type of samples
o Two groups of samples: Control (C) and Test (T)
o Two conditions in each group: A, B
o Three replicates each: CA_1, 2, 3; CB_1, 2, 3; TA_1, 2, 3; TB_1, 2, 3;

Number of gels: 6 (Dye swap labeling)

Data analysis: one way and two way ANOVA

Cy2
Cy3
Cy5

Gel 1
Internal Standard
CA_1
CB_1
Gel 2
Internal Standard
TA_1
TB_1
Gel 3
Internal Standard
CA_2
TA_2
Gel 4
Internal Standard
TB_2
CB_2
Gel 5
Internal Standard
TB_3
CA_3
Gel 6
Internal Standard
CB_3
TA_3
Internal Standard = CA_1, 2, 3; CB_1, 2, 3; TA_1, 2, 3; TB_1, 2, 3

 

2D DIGE Cross-gel Analysis 2D DIGE Cross-gel Analysis
2D DIGE Cross-gel Analysis

Data Report

1. Average protein expression ratio between any two groups

2. Statistical analysis on Control vs. Test

3. Statistical analysis on A vs. B

4. Statistical analysis on the interaction of 2 conditions (2-way ANOVA)

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Study Design 4: Example

 

Type of samples
o Multiple groups of samples: Group 1, 2, 3, 4
o Multiple conditions in each group: Condition A, B, C, D
o Multiple replicates each

Number of gels
o Total number of samples divided by 2
o Dye swap labeling and random pairing

Data analysis: one way and two way ANOVA

2D DIGE Cross-gel Analysis 2D DIGE Cross-gel Analysis
2D DIGE Cross-gel Analysis

Data Report

1. Average protein expression ratio between any two groups

2. Statistical analysis on each condition

3. Statistical analysis on the interactions between any 2 conditions

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