biotin) and then with free biotin(to block all remaining free binding sites on the added avidin). The free biotin is washed away and antibody detection can proceed(Lydan and O'Day, 1991) WASHING Thorough washing is critical to obtaining clean blots, so washing imes and solution volumes should always be generous. It is impor- tant to realize that protein binding and antibody interactions do not all occur at the surface but rather throughout the entire hickness of the membrane. For this reason, thorough soaking and luilibration of the membrane is critical at every step Washing should always be performed at room temperature and with thorough agitation. The exact volume of wash buffer will depend on the container used for washing, but the depth of the solution should be about 1 cm. When protocols call for changing wash solution, this should not be ignored. The higher the sensitiv ity of the detection method, the more important is scrupulous washing technique What Composition of wash Buffer Should You Use Standard wash buffer simply consists of PBs or TBs with added detergent: Tween-20 is routinely used at 0. 1%, although Tween concentrations can be raised to as high as 0.3% to help reduce background. Concentrations higher than this tend to disrupt anti body binding. Triton, NP-40 and SDs should not be used, as they may strip off bound antibodies or target proteins Another method sometimes used to increase the effectiveness of washing is increasing the concentration of salt in the wash solu- tion. High salt reduces charge-mediated effects, which tend to be less specific, and favors hydrophobic interactions, which are more specific. The usual upper limit for NaCl concentration in wash buffers is 500mM. (Standard PBS and TBS contain 130mM NaCl) What Are Common blot size Format. and Handling Techniques? Small blots, or larger blots cut into strips for analysis with several different antibodies, can be incubated in large centrifuge tubes or specialized strip-incubation trays. Larger blots should be incubated in trays. Centrifuge tubes are convenient and allow small reagent volumes to be used. Even with trays, there only needs to be sufficient blocking or antibody solution to submerge Riisbiotin) and then with free biotin (to block all remaining free binding sites on the added avidin). The free biotin is washed away, and antibody detection can proceed (Lydan and O’Day, 1991). WASHING Thorough washing is critical to obtaining clean blots, so washing times and solution volumes should always be generous. It is impor￾tant to realize that protein binding and antibody interactions do not all occur at the surface but rather throughout the entire thickness of the membrane. For this reason, thorough soaking and equilibration of the membrane is critical at every step. Washing should always be performed at room temperature and with thorough agitation. The exact volume of wash buffer will depend on the container used for washing, but the depth of the solution should be about 1cm. When protocols call for changing wash solution, this should not be ignored. The higher the sensitiv￾ity of the detection method, the more important is scrupulous washing technique. What Composition of Wash Buffer Should You Use? Standard wash buffer simply consists of PBS or TBS with added detergent: Tween-20 is routinely used at 0.1%, although Tween concentrations can be raised to as high as 0.3% to help reduce background. Concentrations higher than this tend to disrupt anti￾body binding. Triton, NP-40 and SDS should not be used, as they may strip off bound antibodies or target proteins. Another method sometimes used to increase the effectiveness of washing is increasing the concentration of salt in the wash solu￾tion. High salt reduces charge-mediated effects, which tend to be less specific, and favors hydrophobic interactions, which are more specific. The usual upper limit for NaCl concentration in wash buffers is 500 mM. (Standard PBS and TBS contain 130 mM NaCl.) What Are Common Blot Size, Format, and Handling Techniques? Small blots, or larger blots cut into strips for analysis with several different antibodies, can be incubated in large centrifuge tubes or specialized strip-incubation trays. Larger blots should be incubated in trays. Centrifuge tubes are convenient and allow small reagent volumes to be used. Even with trays, there only needs to be sufficient blocking or antibody solution to submerge 382 Riis