Ability to Strip and Reprobe Radioactive and chemiluminescent systems are ideally suited to stripping and reprobing. Other systems(chemifluorescent and chromogenic) leave insoluble precipitates over the bands of inter- est; these precipitates can be removed only with the use of sol vents, which is an unpleasant extra step and can be hard on blots Not all targets survive this treatment. (See below for important cautions regarding stripping.) Equipment and Facility requirements Radioactivity can be used only after fulfilling stringent training nd licensing requirements. Radioactive methods, like chemilumi nescent methods, also require darkroom facilities(unless storage phosphor equipment is available). Fluorescent methods require specialized scanning equipment. Chromogenic methods do not require any specialized facilities or equipment. What Are the Keys to obtaining High-Quality Results? Careful choice of materials, an understanding of the questio your experiments are intended to answer, and an appreciation of the fact that every new system requires optimization are all neces- sary for obtaining good results. Optimization takes time, but it will pay off in the final result. It is also important to develop consistency in technique from day to day, and to keep detailed and accurate records. Consistency and good record-keeping will make it much asier to isolate the source of any problem that may come up later Which Transfer Membrane Is Most Appropriate to Your needs? The same considerations go into the choice of membrane that go into the choice of any other component of your detection strat egy. What is the molecular weight of your protein? What detec tion method will you use, and does this method have special membrane requirements? Do you intend to strip and reprobo your blots? (See Table 13.2.) itrocellulose wets easily and gives clean backgrounds. Unfo tunately, it is physically fragile(liable to tear and crack), especially when dry. This fragility makes nitrocellulose undesirable for use in stripping and reprobing The problem of physical fragility has been overcome with the introduction of supported nitrocellulose, which has surfaces of nitrocellulose over a core or"web"of phys- ically stronger material. The added physical strength comes at the cost of slightly higher background Western blotting 379Ability to Strip and Reprobe Radioactive and chemiluminescent systems are ideally suited to stripping and reprobing. Other systems (chemifluorescent and chromogenic) leave insoluble precipitates over the bands of inter￾est; these precipitates can be removed only with the use of sol￾vents, which is an unpleasant extra step and can be hard on blots. Not all targets survive this treatment. (See below for important cautions regarding stripping.) Equipment and Facility Requirements Radioactivity can be used only after fulfilling stringent training and licensing requirements. Radioactive methods, like chemilumi￾nescent methods, also require darkroom facilities (unless storage phosphor equipment is available). Fluorescent methods require specialized scanning equipment. Chromogenic methods do not require any specialized facilities or equipment. What Are the Keys to Obtaining High-Quality Results? Careful choice of materials, an understanding of the questions your experiments are intended to answer, and an appreciation of the fact that every new system requires optimization are all neces￾sary for obtaining good results. Optimization takes time, but it will pay off in the final result.It is also important to develop consistency in technique from day to day, and to keep detailed and accurate records. Consistency and good record-keeping will make it much easier to isolate the source of any problem that may come up later. Which Transfer Membrane Is Most Appropriate to Your Needs? The same considerations go into the choice of membrane that go into the choice of any other component of your detection strat￾egy. What is the molecular weight of your protein? What detec￾tion method will you use, and does this method have special membrane requirements? Do you intend to strip and reprobe your blots? (See Table 13.2.) Nitrocellulose wets easily and gives clean backgrounds. Unfor￾tunately, it is physically fragile (liable to tear and crack), especially when dry. This fragility makes nitrocellulose undesirable for use in stripping and reprobing. The problem of physical fragility has been overcome with the introduction of supported nitrocellulose, which has surfaces of nitrocellulose over a core or “web” of phys￾ically stronger material. The added physical strength comes at the cost of slightly higher background. Western Blotting 379