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Cross-gel Analysis

 

 

 

Ideal for comparing more than 3 samples, or multiple replicates in each experimental group

An Internal Standard made up of equal amounts 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 spot statistics
o Allows detection of real 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
Standard
Sample 1 Sample 2
Gel 2
Standard
Sample 3 Sample 4
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

 

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
Standard
C_1
T_1
Gel 2
Standard
T_2
C_2
Gel 3
Standard
C_3
T_3
Gel 4
Standard
T_4
C_4
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

 

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
Standard
CA_1
CB_1
Gel 2
Standard
TA_1
TB_1
Gel 3
Standard
CA_2
TA_2
Gel 4
Standard
TB_2
CB_2
Gel 5
Standard
TB_3
CA_3
Gel 6
Standard
CB_3
TA_3
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)

 

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