Quantitative Dot-Immunoblot Assay to Measure Protein in Multiple Brain Samples

Western blotting (1) is frequently used to detect changes in the amount of protein in tissue after injury or with disease, particularly if the concentration of the protein is low. However, this technique has several limitations. The procedure is time-consuming and only a few samples can be assayed at any given time. Furthermore, Western blotting is subject to variations in protein loading and requires electrophoretic transfer, factors which limit the ability to make quantitative estimates of the amount of protein in the sample. In dotimmunoblotting, many samples can be assayed simultaneously, and protein transfer is not necessary, since the sample is directly applied to the nitrocellulose membrane (2). We modified the quantitative dot-immunoblot technique described by Jahn et al. (3), improving the reproducibility and reliability by which measurement of the amount of protein in tissue samples can be made. In this assay, purified protein is used to generate a standard curve and this curve is used to determine the absolute amount of protein present in the tissue homogenates. Detection is by direct counting of radioactivity rather than by densitometric analysis. By minimizing adsorption of protein to plastic, by improving the application of the sample to nitrocellulose, by correcting for nonspecific binding in crude homogenates and by directly counting radioactivity, nanogram quantities of protein can be reliably detected in tissue samples. We describe the use of this assay to measure the amount of neurofilament (NF) 200-kDa protein in different regions of the brain in rabbits subjected to cerebral hypoxia-ischemia and reperfusion. To inflict brain injury, anesthetized, immature male rabbits were subjected to cerebral hypoxia-ischemia and reperfusion by using a recently developed model (4). Rabbits first breathed 2.5% oxygen for 8 min and then 8 min of cerebral ischemia was induced by rais-