Reverse Phase Protein Arrays Based Protein Expression Analysis: A Procedure for Simultaneous Quantification of Expression of Multiple Proteins from Cell Lysate
Trascrizione
A tumor sample contains a heterogeneous population of proteins. Using tumor lysate, multiple such proteins can be analyzed parallelly to identify the protein of interest. This high-throughput technique is called Reverse Phase Protein Arrays or RPPA.
To perform RPPA, begin by taking an immunoassay glass slide coated with nitrocellulose. Spot an array of tumor lysate proteins on the slide. Incubate to allow protein capture on nitrocellulose. Subsequently, treat the slide with a blocking buffer that blocks non-specific antibody binding sites on proteins.
Remove the excess blocking solution. Now, mount the protein-coated slide in a holder and seal it tightly. This creates temporary wells over the slide surface. Next, load the wells with a primary antibody solution. These antibodies recognize epitopes present on the corresponding target from a mixture of proteins on the array.
Finally, add fluorescently-labeled secondary antibodies. These antibodies attach to the pre-bound primary antibodies forming a complex. Wash the slide to remove any unbound secondary antibodies. Unfasten the slide from the holder and allow it to air dry.
Scan the slide using a fluorescent scanner. The antibody-tagged target proteins fluoresce at a specific wavelength and appear as colored spots on the slide surface.
