This chapter describes the role of Western Blotting in determining antibody specificity. The procedure is illustrated using whole protein extracts obtained from non-cultured human keratinocytes and the bladder TCC cell line RT4. Proteins are separated by two-dimensional (2D) gel electrophoresis blotted to a nitrocellulose membrane, and developed using the HRP color development reagent and/or the ECL procedure. Place the wet nitrocellulose membrane on top of the gel. The operation should be done under the buffer. Rub the membrane from one end to the other to eliminate bubbles. Primary antibodies should be diluted to a volume of 8 ml. The dilution should be optimized in preliminary experiments for the best results in terms of high signal and low background. Cut the appropriate area of the blot with a scalpel using the X-ray film as reference and wet by capillarity in the skimmed milk solution. Incubate with shaking for 1 h at room temperature or overnight in the cold room in the same solution. © 2006