TISSUE LYSATE MICROARRAYS
Tissue Lysate Microarrays (or Reverse Phase Protein Arrays, RPPA) consist of very small amounts of cell or tissue lysates that are spotted into a microarray and probed for the presence of target proteins. The lysates may be generated from cellular lysates, from cultured cells or laser-capture microdissected cells, body fluids such as serum, CSF, urine, vitreous, and saliva. The proteins from the lysates are immobilized in an array of small spots (150-300 um) that are then incubated with a single specific antibody to detect expression of the target protein across many samples. Multiple proteins may be probed for by labeling the detection antibodies with fluorescent compounds with different emission spectra. Compared to histological examination of tissue slices, RPPA arrays with tissue lysates provide a high-throughput quantitative assessment of protein expression in comparative studies and tumor biology (Charboneau et al., 2002), with potentially thousands of samples examined in one array. Reverse Phase Arrays were first developed to examine the progression of cancer tumors in individual patients, and the role of protein expression in cell signaling pathways. Subsequently, the use of RPPA has been applied to vaccine development, using pathogen proteins and interrogating them with serum from vaccinated subjects (Davies et al, 2004), as well as pharmacological studies of tumor responses to drugs (Wallqvist et al., 2002), and examining protein expression profiles of disease in animal models.
Porous nitrocellulose film (PNC) is the substrate of choice for RPPA largely because of its high protein binding capacity, which can exceed non-porous 2-dimensional substrates such as functionalized glass by 500X. Because proteins of interest are often expressed at low levels relative to ‘housekeeping’ proteins, the high binding capacity of the 3-dimensional PNC enables sensitive detection of rare proteins, and detection of small changes in protein expression of <1.5% (Anderson et al., 2011). Multiple protocols based on nitrocellulose film slides exist for RPPA assays, directed at detection of low-abundance proteins in tissue lysates. At Grace Bio-Labs we have developed reagents and protocols designed to obtain consistent and sensitive results with our nitrocellulose film.
Use of Grace Bio-Labs products in Tissue Lysate microarrays:
As mentioned above, porous nitrocellulose is the substrate of choice for RPPA. ONCYTE® PNC (Porous NitroCellulose) Microarray Slides from Grace Bio-Labs have been shown to bind up to 500x more protein in a given spot than planar microarray substrates, resulting in a much higher capacity to capture analyte from sample solution. Further, the non-covalent nature of protein binding to ONCYTE Nitrocellulose film slides and the 3-dimensional support matrix results in a protein-friendly environment, allowing maintenance of native protein structure and retention of structure dependent binding activity. ONCYTE Nitrocellulose film slides and Grace Bio-Labs reagents are designed to provide a complete system for sensitive and consistent results for your protein arrays. Our film slides are manufactured with a stringent quality control protocol that ensures minimal variability and consistently low fluorescence background compared to other commercially available film slides.
Incubation chambers from Grace Bio-Labs, including the HybriSlip and ProPlate, provide excellent results with all protein array applications. The incubation chamber is an important parameter which is often overlooked when performing microarray experiments. The ideal chamber should allow for sufficient sample mixing during the assay incubation and wash steps and should minimize the volume of sample required. Active mixing has been shown to significantly affect assay signal and uniformity.
Coverslips generally allow for the lowest sample incubation volumes and, but do not allow sample mixing during incubation. If the use of a coverslip is necessary for your particular assay, we recommend the use of Grace Bio-Labs HybriSlip™ over conventional glass coverslips. In addition, Grace Bio-Labs has developed incubation chambers which facilitate incubations for a wide range of sample volumes with various The Pro-Plate® chamber from Grace Bio-labs is excellent for most microarray applications on slide or plate format.
Charboneau et al., Utility of reverse phase protein arrays: Applications to signaling pathways and human body arrays. Briefings in Functional Genomics and Proteomics 2002, 3:305.
Davies et al., Profiling the humoral immune response to infection by using proteome microarrays: High-throughput vaccine and diagnostic antigen discovery
Wallqvist et al., Establishing Connections between microarray expression data and chemotherapeutic cancer pharmacology. Molecular Cancer Therapeutics, 2002: 1:311.
Anderson et al., High resolution mapping of the cardiac transmural proteome using reverse phase protein microarrays. Molecular & Cellular Proteomics, 2011: 10:7.