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

 

	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

 

	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

 

	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

	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