Post on 08-Feb-2016
description
Protein Lysate Microarrays
Clay ScottRyan McConnellShannon Neeley
Biological and Technical Background
Motivation
DNA/RNA microarrays are used to determine gene expression
Proteomic profiling can help yield more direct answers to biological questions
Those molecules that can answer our questions are proteins, not mRNA
– Biological effector molecules– Diagnostic markers– Pharmaceutical targets
History of Proteomic Profiling
2D-PAGE (two-dimensional polyacrylamide gel electrophoresis)– Introduced in 1975– Semiquantitation of most abundant 1000 spots– Problems with identifying spots with a particular protein
Microarray formats for proteomic profiling– More recent development (papers published in 2001)– Robotic spotting of antibodies that capture the protein
molecule to be assessed
Reverse-phase protein lysate microarrays
Opposite configuration from previous microarray
Samples assessed are robotically spotted An antibody is used to measure amount of a
particular protein present in the sample Limitation: Measure one protein per slide Advantage: All samples analyzed side-by-side
in a single array – Can compare protein levels across samples rather
than samples across protein type
Protein lysate preparation
Lysis: The dissolution or destruction of cells by the action of a specific lysin that disrupts the cell membrane
Lysate: The cellular debris and fluid produced by lysis
Lysis Method Description ApparatusWaring BlenderPolytronDounce HomogenizerPotter-Elvehjem HomogenizerFrench Press
Sonication SonicatorHigh frequency sound waves shear cells
Freeze/ThawFreezer or dry ice/ethanol
Repeated cycles of freezing and thawing disrupt cells through ice crystal formation
Manual grinding Mortar and pestleGrinding plant tissue, frozen in liquid nitrogen
Table 1. Techniques used for the physical disruption of cells.
MechanicalRotating blades grind and disperse cells and tissues
Liquid Homogenization
Cell or tissue suspensions are sheared by forcing them through a narrow space
Protein Lysate Array Design
Each microarray is a glass slide containing particular protein samples from different patients
18 spots for every sample– 3 replicates– 6 dilution levels (with
dilution factor of 2)
Western Blotting—used to screen specificity of antibodies
Choose Antibody that will bind to protein
Detection of protein on microarray
The slide is exposed to the antibody Antibody binds to the protein, depending on
how much protein is present Microarray is scanned to form an image with
darker spots reflecting higher levels of protein Use two antibody detection system
Statistical Applications
From Image to Number
Software draws a circle around each spot
Darker spots have greater quantities of protein
sVOL reflects the total amount of protein
sVOL = (πr2)[(average intensity inside circle)-(average background intensity)]
Two-fold serial dilutions
The Data Set 80 x 6 x 3 matrix of sVOL values for the caspase protein 80 patients on one microarray chip 6 two-fold serial dilutions for each patient 3 replicates for each patient Control serum data set for calibration and error-assessment purposes
DilutionsP
A
T
I
E
N
T
S
1
80
6
Potential Tasks to Undertake
1. From the 18 sVOL values for each sample, extract a single robust number that is representative of that sample
° for example, the mean is not robust
2. Provide an error estimate of that number
Saturation DetectionDevise procedure for extracting linear portion of log(sVOL)
For highly concentrated samples, circles drawn by computer may be too small (sVOL smaller than expected)
For large dilutions, sVOL is dominated by background noise (sVOL tails off)
Fine Tuning the Segmentation Program
Outlying sVOL values may occur even when saturation is not a problem
Detecting outliers may help improve the segmentation software