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 1 | More than three individual samples |
| Study Design 2 | Two groups of samples, multiple replicates per group |
| Study Design 3 | Two groups of samples, two conditions per group, multiple replicates |
| Study Design 4 | Multiple 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
| Cy2 | Cy3 | Cy5 | |
| Gel 1 | Internal Standard | Sample 1 | Sample 2 |
| Gel 2 | Internal Standard | Sample 3 | Sample 4 |
Internal Standard = Sample 1-4
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
| 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
Data Report
- Average protein expression ratio between
control and test group - 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
| 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
Data Report
- Average protein expression ratio between any two groups
- Statistical analysis on Control vs. Test
- Statistical analysis on A vs. B
- 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
Data Report
- Average protein expression ratio between any two groups
- Statistical analysis on each condition
- Statistical analysis on the interactions between any 2 conditions