- Coomassie Stains & Protein Gel Documentation
- Nucleic Acid Gel Documentation
- ELISA
- EMSA/Gel Shift Assay
- Glycoprotein Detection
- In-Cell Western™ Assay
- In-Gel Western Assay
- Microwestern Array
- Northern Blot
- On-Cell Western Assay
- Protease Assay
- Protein Array
- Quantitative Western Blot
- Reporter Gene Assay
- Reverse Phase (Lysate) Array
- RNAi Analysis
- Small Animal Imaging
- Southern Blot
- Tissue Section Imaging
- Transcription Factor Assay
Email bio-eu@licor.com
APPLICATIONS for the
ODYSSEY® CLx
Applications for the Odyssey CLx Infrared Imaging System
REVERSE PHASE (LYSATE) ARRAY
Applications Overview
The protein array is a higher-throughput way to generate information about protein abundance or modification state.
Using IR fluorescence to detect your protein arrays will give you
Dramatically reduced background autofluorescence of nitrocellulose-coated slides1
Sensitivity in the femtogram range2
Wide dynamic range
Multiplexed detection to quantify and compare two targets3
Simplified detection protocol3
Lysate (reverse phase) arrays contain complex samples, such as cell or tissue lysates, that are printed on an array surface and interrogated with antibodies4. Advantages include:
More quantitative and reproducible than Westerns (intra-chip variation ˜0.1%2)
Wide dynamic range
Conserve precious samples
Very sensitive – can detect femtogram or single-cell protein levels2
Spot recombinant protein standards for absolute quantification2
Run many replicates and dilutions easily
[ABOVE] Figure 2. Reproducibility of ERK detection using infrared dyes
Lysate was prepared from a human breast cancer tissue sample. It was arrayed in triplicate in a series of two-fold dilutions, and stained with rabbit anti-ERK primary Ab and IRDye 800CW dye-labeled goat anti-rabbit. Linearity and reproducibility of this data is shown in the graph. The average of three replicates is plotted; error bars are included but are very small and difficult to see. Adapted from Calvert, VS et al. Clin Prot J. 1(1):81-89 (2004).
[LEFT] Figure 3. Multiplexed detection of phosphorylated and total ERK protein levels.
Tissue samples were obtained for three human breast cancer patients; lysates were prepared from these and from a Jurkat T cell control. The array was probed simultaneously with antibodies against phospho-ERK (mouse) and total ERK (rabbit), and detected with near-infrared labeled secondary antibodies (red, green). Overlaid image of total and phospho-ERK levels is shown at the top (red + green = yellow). Adapted from Calvert, VS et al.
Clin Prot J. 1(1):81-89 (2004).
1. Sheehan, KM et al. Mol Cell Proteomics. 4(4): 346-55 (2005)
2.Loebke, C et al. Proteomics. 7(4):558-64(2007)
3.Ambroz, K. et al. Poster presentation, Chips to Hit Annual Meeting (2005)
4.Hall, DA et al. Mech Ageing Dev. 128(1):161-7 (2007)
Workflow
[LEFT] Schematic protocol for near-infrared lysate (reverse phase) arrays. Adapted from Calvert, VS et al. Clin Prot J. 1(1):81-89 (2004).
[LEFT] Schematic of lysate protein array concept. Adapted from Sheehan, KM et al.
Mol Cell Proteomics. 4(4): 346-55 (2005)
Technical Resources
Tips for Antibody and Lysate Arraying LI-COR Biosciences (2005)
Click here for a complete list of protocols.
PubMed
Ambroz, KL et al.
Blocking and detection chemistries affect antibody performance on reverse phase protein arrays.
Proteomics. 8(12):2379-83 (2008)Pipinos II et al.
Chronically ischemic mouse skeletal muscle exhibits myopathy in association with mitochondrial dysfunction and oxidative damage.
Am J Physiol Regul Integr Comp Physiol. 295(1):R290-6(2008)Loebke, C et al.
Infrared-based protein detection arrays for quantitative proteomics.
Proteomics. 7(4):558-64(2007)Sahin, O et al.
Combinatorial RNAi for quantitative protein network analysis.
Proc Natl Acad Sci USA. 104(16):6579-84(2007)Calvert, VS et al.
Development of Multiplexed Protein Profiling and Detection Using Near Infrared Detection of Reverse-Phase Protein Microarrays.
Clin Prot J. 1(1):81-89 (2004)
