2D DIGE Data Analysis: Cross Gel

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

An “Internal Standard” containing equal amounts of protein of each sample is run on all gels.

Importance of the Internal Standard

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

How many replicates do I need?

  • Cell lines: 4
  • Mice: 4+
  • Humans: more are better within the constraints of reality

Choose from four study designs:

Study Designs
Study Design 1More than three individual samples
Study Design 2Two groups of samples, multiple replicates per group
Study Design 3Two groups of samples, two conditions per group, multiple replicates
Study Design 4Multiple groups of samples, multiple conditions per group, multiple replicates

Study Design 1: Example

Type of samples

  • Four individual samples: sample 1, 2, 3, 4
  • No replicates

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

Cy2Cy3Cy5
Gel 1Internal StandardSample 1Sample 2
Gel 2Internal StandardSample 3Sample 4

Internal Standard = Sample 1-4

2D DIGE cross-gel data analysis for study design 1: Spot map showing the selected spot aligned on different gels
2D DIGE cross-gel data analysis for study design 1: protein ratios of the selected spot among 4 samples
2D DIGE cross-gel data analysis for study design 1: 3D view of the selected spot on different gels

Data Report

Protein expression ratio between any 2 samples

Study Design 2: Example

Type of samples

  • Two groups of samples: Control (C) and Test (T)
  • 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

Cy2Cy3Cy5
Gel 1Internal StandardC_1T_1
Gel 2Internal StandardT_2C_2
Gel 3Internal StandardC_3T_3
Gel 4Internal StandardT_4C_4

Internal Standard = C1-4, T1-4

2D DIGE cross-gel data analysis for study design 2: Spot map showing the selected spot aligned on different gels
2D DIGE cross-gel data analysis for study design 2: average protein ratios of the selected spot between Control and Test group (n=4)
2D DIGE cross-gel data analysis for study design 2: 3D view of the selected spot on different gels

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

  • Two groups of samples: Control (C) and Test (T)
  • Two conditions in each group: A, B
  • 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

Cy2Cy3Cy5
Gel 1Internal StandardCA_1CB_1
Gel 2Internal StandardTA_1TB_1
Gel 3Internal StandardCA_2TA_2
Gel 4Internal StandardTB_2CB_3
Gel 5Internal StandardTB_3CA_3
Gel 6Internal StandardCB_3TA_3

Internal Standard = CA_1, 2, 3; CB_1, 2, 3; TA_1, 2, 3; TB_1, 2, 3

2D DIGE cross-gel data analysis for study design 3: Spot map showing the selected spot aligned on different gels
2D DIGE cross-gel data analysis for study design 3: protein ratios and statistical analysis of the selected spot between any two groups
2D DIGE cross-gel data analysis for study design 3: 3D view of the selected spot on different gels

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

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

Number of gels

  • Total number of samples divided by 2
  • Dye swap labeling and random pairing

Data analysis: one way and two way ANOVA

2D DIGE cross-gel data analysis for study design 4: Spot map showing the selected spot aligned on different gels
2D DIGE cross-gel data analysis for study design 4: protein ratios and statistical analysis of the selected spot between any two groups
2D DIGE cross-gel data analysis for study design 4: 3D view of the selected spot on different gels

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