Reverse Phase Protein Lysate Microarray

Reverse Phase Protein Lysate Microarray

Reverse phase protein array (RPPA) is a protein array designed as a micro- or nano-scaled dot-blot platform that allows measurement of protein expression levels in a large number of biological samples simultaneously in a quantitative manner when high-quality antibodies are available. Technically, minuscule amounts of a) cellular lysates, from intact cells or laser capture microdissected cells, b)body fluids such as serum, CSF, urine, vitreous, saliva, etc., are immobilized on individual spots on a microarray that is then incubated with a single specific antibody to detect expression of the target protein across many samples. A summary video of RPPA is available One microarray, depending on the design, can accommodate hundreds to thousands of samples that are printed in a series of replicates. Detection is performed using either a primary or a secondary labeled antibody by chemiluminescent, fluorescent or colorimetric assays. The array is then imaged and the obtained data is quantified. Multiplexing is achieved by probing multiple arrays spotted with the same lysate with different antibodies simultaneously and can be implemented as a quantitative calibrated assay. In addition, since RPMA can utilize whole-cell or undissected or microdissected cell lysates, it can provide direct quantifiable information concerning post translationally modified proteins that are not accessible with other high-throughput techniques. Thus, RPMA provides high dimensional proteomic data in a high throughput, sensitive and quantitative manner. However, since the signal generated by RPMA could be generated from unspecific primary or secondary antibody binding, as is seen in other techniques such as ELISA, or immunohistochemistry, the signal from a single spot could be due to cross-reactivity. Thus, the antibodies used in RPMA must be carefully validated for specificity and performance against cell lysates by western blot.

RPMA has various uses such as quantitative analysis of protein expression in cancer cells, body fluids or tissues for biomarker profiling, cell signaling analysis and clinical prognosis, diagnosis or therapeutic prediction. This is possible as a RPMA with lysates from different cell lines and or laser capture microdissected tissue biopsies of different disease stages from various organs of one or many patients can be constructed for determination of relative or absolute abundance or differential expression of a protein marker level in a single experiment. It is also used for monitoring protein dynamics in response to various stimuli or doses of drugs at multiple time points. Some other applications that RPMA is used for include exploring and mapping protein signaling pathways, evaluating molecular drug targets and understanding a candidate drug’s mechanism of action. It has been also suggested as a potential early screen test in cancer patients to facilitate or guide therapeutic decision making.

Other protein microarrays include forward protein microarrays (PMAs) and antibody microarrays (AMAs). PMAs immobilize individual purified and sometimes denatured recombinant proteins on the microarray that are screened by antibodies and other small compounds. AMAs immobilize antibodies that capture analytes from the sample applied on the microarray . The target protein is detected either by direct labeling or a secondary labeled antibody against a different epitope on the analyte target protein (sandwich approach). Both PMAs and AMAs can be classified as forward phase arrays as they involve immobilization of a bait to capture an analyte. In forward phase arrays, each array is incubated with one test sample such as a cellular lysate or a patient’s serum, but multiple analytes in the sample are tested simultaneously . Figure 1 shows a forward (using antibody as a bait in here) and reverse phase protein microarray at the molecular level.