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Background
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Total cell Iysates are useful as both positive and negative controls in immunoblotting. A431, a human epidermoid carcinoma cell line, and the EGF-stimulated A431 Iysates are used as negative and positive controls, respectively, when studying the phosphorylation cascade initiated by ligand binding to receptor tyrosine kinases. Both A431 and normal human fibroblasts are valuable positive controls for antibodies to proteins expressed in these cells. The mouse fibroblast cell line 3T3 and its RSV-transformed counterpart serve as negative and positive controls, respectively, for antibodies to proteins that are phosphorylated as a result of transformation by RSV (Rous sarcoma virus). Madin-Darby bovine kidney (MDBK) cell Iysate displays proteins expressed in bovine kidney and PC12 (rat phaeochromocytoma) cell Iysate can be expected to mirror the protein expression of cells and tissues of neuroectodermal origin. Likewise, Jurkat cells express the proteins unique to human T-cells. Rat brain is a widely used positive control for those proteins that are exclusively expressed in brain. At right is an example of the use of positive and negative control Iysates in western blotting. The blot of 3T3 cell Iysate (lane 1) and RSV-transformed 3T3 (lane 2) was probed with anti-phosphotyrosine peroxidase conjugate to reveal those proteins that are tyrosine-phosphorylated in response to transformation by Rous sarcoma virus.
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