Module 2: Systems Engineering (M2D6)

Transcription

1 Module 2: Systems Engineering (M2D6) Reminder of goals of Module 2 Scale it up! (For real this time!) Densitometry -- how to analyze your WB data. M2D7 -- to robot or not to robot...

2 Module 2 overview: Erlotinib EGFR mutant? PI(4,5)P2 PI(3,4,5)P3 PI3K Akt Grb2 Sos P STAT3 Ras P Erk1/2 P STAT3 P STAT3?

3 Module 2 overview: Erlotinib EGFR mutant? PI(4,5)P2 PI(3,4,5)P3 PI3K Akt Mutation Analysis Grb2 Sos P STAT3 P Erk1/2 Ras P STAT3 P STAT3? WB Analysis Viability Analysis

4 Module 2 overview: Erlotinib EGFR mutant? PI(4,5)P2 PI(3,4,5)P3 PI3K Akt Mutation Analysis Erlotinib WT EGFR PI(4,5)P2 PI(3,4,5)P3 PI3K Akt Grb2 Sos P STAT3 Grb2 Sos P STAT3 P Erk1/2 Ras P STAT3 P STAT3? WB Analysis P Erk1/2 Ras P STAT3 P STAT3 Viability Analysis

5 Semi-quantitative analysis: Your experiment 1. All: py1068-egfr & Total EGFR 2. Pathway specific: perk & Total ERK pakt & Total Akt pstat3 & Total STAT3 *Last two + GAPDH

6 What if we wanted a broader network view? Regulatory networks sensitive to tyrosine kinase inhibitors in H3255 and MKN45 cells revealed by PhosphpScan-SILAC study by National Academy of Sciences Guo A et al. PNAS 2008;105:

7 Semi-quantitative analysis: Protein Microarray A few signals per spot -- up to hundreds of spots per slide.

8 Semi-quantitative analysis: Protein Microarray Strength: Multiplex -- many conditions can be screened Weakness: Non-specific interactions (also huge problem in WB) A few signals per spot -- up to hundreds of spots per slide.

9 Quantitative analysis: ELISA Detection Signal Capture Antibody Biotin Detection Antibody Capture Antibody One signal per well - but up to 384 wells/experiment.

10 Quantitative analysis: ELISA Detection Signal Capture Antibody Biotin Detection Antibody Capture Antibody One signal per well - but up to 384 wells/experiment.

11 Quantitative analysis: ELISA Capture Antibody Detection Signal Detect with anti-phospho Biotin antibody Detection Antibody Capture Antibody Most common to capture total protein: phosphorylated + unphosphorylated One signal per well - but up to 384 wells/experiment.

12 Quantitative analysis: ELISA How to quantify: ELISA Standard Curve #2 4 3 Very often not linear relationships Abs Log 10 [protein], pg Y=Bottom + (Top-Bottom)/(1+10^((LogEC50-X)*HillSlope)) General protocol: Create a standard curve on each plate (usually supplied with kits or make your own)

13 Quantitative analysis: Multiplexed bead based ELISA

14 Quantitative analysis: Multiplexed bead based ELISA

15 Quantitative analysis: Multiplexed bead based ELISA

16 Quantitative analysis: Multiplexed bead based ELISA

17 Quantitative analysis: Multiplexed bead based ELISA Theoretically up to hundreds of conditions per well (sometimes >1500!) wells per experiment In reality: different phosphoproteins / well max

18 Semi-quantitative analysis: Western blot EGF (ng/ml) 250 kda py1068-egfr Total EGFR GAPDH Rabbit Ab Goat Ab 25 Infrared Secondary Abs: 250 kda py1068-egfr anti-rabbit IR680 anti-goat IR Total EGFR SKOV3 A few signals per lane -- up to ~17 lanes per mini gel.

19 Densitometry: Step1: Separate Channels & Invert

20 Densitometry: Step2: Choose regions of interest This is what it looks like in ImageJ

21 Densitometry: Step3: Plot pixel intensity (line drawn vertically through box) This is what it looks like in ImageJ

22 Step 4: Define area to analyze Warning: Can be very subjective! Step 5: Use magic wand to calculate area under the curve Step 6: Keep track of what value corresponds to each experimental condition

23 Step 4: Define area to analyze Warning: Can be very subjective! Step 5: Use magic wand to calculate area under the curve Step 6: Keep track of what value corresponds to each experimental condition Total pixel intensity

24 Semi-quantitative analysis: Western blot 250 kda kda EGF (ng/ml) py1068-egfr Total EGFR GAPDH py1068-egfr (py1068-egfr)/(egfr) Densitometric Analysis GAPDH EGF Concentration (ng/ml) Total EGFR Relative pixel intensity SKOV3

25 Module 2: Systems Engineering (M2D6) Reminder of goals of Module 2 Scale it up! (For real this time!) Densitometry -- how to analyze your WB data. M2D7 -- to robot or not to robot...