digital raw data from fluorescence scanners (and storag phosphor scanners for radioactive detection) is inherently quan titative. The linear range of film-based systems (limited by the response of the film)is a little better than one order of magnitude hile the manufacturers of fluorescent scanners claim something closer to two orders of magnitude There are several cautions to bear in mind when considering protein blot quantitation Standards(known amounts of purified target protein-not to be confused with molecular weight stan dards)must be run on every blot, since even with the most con- stent technique there can be blot-to-blot variation. The standard should be loaded on the gel, electrophoresed, and transferred in exactly the same way your samples are The determination of quantity can only be made within the ange of standards on the blot: extrapolation beyond the actual standard values is not valid. This together with the limited linear range means that several dilutions of the unknown sample usually must be run on the same blot given all the lanes of standards and sample dilutions, the amount of quantitative data that can be extracted from a single blot is somewhat limited. Protein blot uantitation can be useful, but it is not a substitute for techniques such as elisa or ria Antibody requirements Typically the choice of available primary antibodies is not as vide as that of the other elements of the detection system. Primary antibodies can be obtained from commercial suppliers, non-profit repositories, and even other researchers. Tracking down a primary antibody can be time-consuming, but publications such as Lin- scottsDirectory(linscott,1999,andhttp://www.linscottsdirec tory. com/index2hmD,the“ Antibody Resource Page”(htp∥ www.antibodyresource.com),theUsenetnewsgroup"methods and Reagents"(bionet. molbio. methds-reagnts ), and Stefan Dubel's recombinantantibodypage(www.mgen.uni-heidelberg.de/sd/ SdscFvsite.htmlandwww.antibody.comcanhelp If no antibodies against your target protein exist, your only options are to raise the antibody yourself or to have someone do it. Companies such as Berkeley Antibody Company, Genosys, Rockland, and Zymed(among many others) can do his kind of work on a contract basis. Whichever route you choose, this is a time-consuming and potentially expensive undertaking 378 Riis
digital raw data from fluorescence scanners (and storagephosphor scanners for radioactive detection) is inherently quantitative. The linear range of film-based systems (limited by the response of the film) is a little better than one order of magnitude, while the manufacturers of fluorescent scanners claim something closer to two orders of magnitude. There are several cautions to bear in mind when considering protein blot quantitation. Standards (known amounts of purified target protein—not to be confused with molecular weight standards) must be run on every blot, since even with the most consistent technique there can be blot-to-blot variation. The standard should be loaded on the gel, electrophoresed, and transferred in exactly the same way your samples are. The determination of quantity can only be made within the range of standards on the blot: extrapolation beyond the actual standard values is not valid. This together with the limited linear range means that several dilutions of the unknown sample usually must be run on the same blot. Given all the lanes of standards and sample dilutions, the amount of quantitative data that can be extracted from a single blot is somewhat limited. Protein blot quantitation can be useful, but it is not a substitute for techniques such as ELISA or RIA. Antibody Requirements Typically the choice of available primary antibodies is not as wide as that of the other elements of the detection system. Primary antibodies can be obtained from commercial suppliers, non-profit repositories, and even other researchers. Tracking down a primary antibody can be time-consuming, but publications such as Linscott’s Directory (Linscott, 1999, and http://www.linscottsdirectory.com/index2.html), the “Antibody Resource Page” (http:// www.antibodyresource.com), the Usenet newsgroup “Methods and Reagents” (bionet.molbio.methds-reagnts), and Stefan Dubel’s recombinant antibody page (www.mgen.uni-heidelberg.de/SD/ SDscFvSite.html) and www.antibody.com can help. If no antibodies against your target protein exist, your only options are to raise the antibody yourself or to have someone else do it. Companies such as Berkeley Antibody Company, Genosys, Rockland, and Zymed (among many others) can do this kind of work on a contract basis. Whichever route you choose, this is a time-consuming and potentially expensive undertaking. 378 Riis
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 379
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 interest; these precipitates can be removed only with the use of solvents, 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 chemiluminescent 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 necessary 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 strategy. What is the molecular weight of your protein? What detection 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. Unfortunately, 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 physically stronger material. The added physical strength comes at the cost of slightly higher background. Western Blotting 379
Table 3.2 Characteristics of transfer membranes Characteristics Nitrocellulose Low background. Easy to block Supported Binding properties similar to nitrocellulose. Higher background than pure nitrocellulose Physically strong Physically strong Highly hydrophobic: requires methanol pre-wetting and dries easily. Good for stripping and reprobir PVDF (polyvinylidene difluoride) membranes are physically ger and have higher protein-binding capacity than lulose. However, they are highly hydrophobic: so much so that hey need to be pre-wetted with methanol before they can be equilibrated with aqueous buffer. When handling PVDF, you should take special care to ensure the membrane does not dry out, since uneven blocking, antibody incubation, washing, or detection can result. If the membrane does dry out, it should be re-equilibrated in methanol and then in aqueous buffer. The high affinity of PVDF for protein gives efficient transfer and high detection efficiency, but it can make background control more difficult. PVDF is the membrane of choice for stripping and reprobing Transfer membranes are available in several pore sizes. The standard pore size, suitable for most applications, is 0. 45 micron Membranes are also commonly available in 0.2 and even 0.1 micron pore size: these smaller pore sizes are suitable for transfer of lower molecular weight proteins, below about 12kDa Transfer efficiency is not good with membranes with a pore size of less than 0.1 micron BLOCKING All transfer membranes have a high affinity for protein. The purpose of blocking is simply to prevent indiscriminate binding of ne detection antibodies by saturating all the remaining binding capacity of the membrane with some irrelevant protein (For a detailed discussion see amersham, n.d. from which much of the following is drawn.) Riis
PVDF (polyvinylidene difluoride) membranes are physically stronger and have higher protein-binding capacity than nitrocellulose. However, they are highly hydrophobic: so much so that they need to be pre-wetted with methanol before they can be equilibrated with aqueous buffer. When handling PVDF, you should take special care to ensure the membrane does not dry out, since uneven blocking, antibody incubation, washing, or detection can result. If the membrane does dry out, it should be re-equilibrated in methanol and then in aqueous buffer. The high affinity of PVDF for protein gives efficient transfer and high detection efficiency, but it can make background control more difficult. PVDF is the membrane of choice for stripping and reprobing. Transfer membranes are available in several pore sizes. The standard pore size, suitable for most applications, is 0.45 micron. Membranes are also commonly available in 0.2 and even 0.1 micron pore size: these smaller pore sizes are suitable for transfer of lower molecular weight proteins, below about 12 kDa. Transfer efficiency is not good with membranes with a pore size of less than 0.1 micron. BLOCKING All transfer membranes have a high affinity for protein. The purpose of blocking is simply to prevent indiscriminate binding of the detection antibodies by saturating all the remaining binding capacity of the membrane with some irrelevant protein. (For a detailed discussion, see Amersham, n.d., from which much of the following is drawn.) 380 Riis Table 13.2 Characteristics of Transfer Membranes Membrane Characteristics Nitrocellulose Low background. Easy to block. Physically fragile. Supported Binding properties similar to nitrocellulose. nitrocellulose Higher background than pure nitrocellulose. Physically strong. PVDF High protein binding capacity. Physically strong. Highly hydrophobic: requires methanol pre-wetting and dries easily. Good for stripping and reprobing
Which Blocking Agent Best Meets Your Needs? The protein most commonly used for the purpose is nonfat dry milk. often referred to as "blotto. used at 0.5% in Pbs contain ing 0.1%Tween-20. Any grocery-store brand of nonfat dry milk can be used Gelatin is isolated from a number of species, but fish skin gelatin is usually considered the best for Western blotting. Fish gelatin is usually used at a concentration of 2%. It is an effective blocker ll not gel at this concentration at 4oC. Bovine serum albumin(BSa)is available in a wide range of grades. Usually a blotting or immunological grade of BsA is appropriate. It is less expensive than fish skin gelatin, and can be used at 2% Normal serum(fetal calf or horse) is used sometimes, at a con- centration of 10%. It can be an effective blocking agent, but is quite expensive. Since serum contains immunoglobulins, it is not compatible with Protein A and some secondary antibodies. Casein can be used at 1%, but it is very difficult to get dry casein into solution. Casein and casein hydrolysate are the basis of some commercial blocking agents Different primary antibodies work better with different block ing agents: nonfat dry milk is usually a good first choice, but when setting up a new method, it is a good idea to evaluate different blockers It has been claimed that some blocking agents, nonfat dry milk particular, can hide or"mask"certain antigens Of course, there must be no component of the blocking agent that the primary or secondary antibodies can specifically react with Some researchers include a second blocking step prior to sec- ondary antibody incubation. However, if the initial blocking is suf ficient and reagent dilutions are optimal, this should not be A more specific kind of blocking may be needed when avidin or streptavidin is used as a detection reagent and the sample con tains biotin-bearing proteins Because of this"endogenous biotin the avidin or streptavidin will pick up these undesired proteins directly. If you suspect this may be a problem, a control reaction can be run with no primary antibody but with the avidin or strep- tavidin detection. The presence of bands in this control reaction will indicate that the avidin or streptavidin is binding to the endogenous biotin The remedy for such a situation is to treat the blot prior to anti- dy incubation first with avidin (to bind all the endogenous Western blotting 38
Which Blocking Agent Best Meets Your Needs? The protein most commonly used for the purpose is nonfat dry milk, often referred to as “blotto,” used at 0.5% in PBS containing 0.1% Tween-20. Any grocery-store brand of nonfat dry milk can be used. Gelatin is isolated from a number of species, but fish skin gelatin is usually considered the best for Western blotting. Fish gelatin is usually used at a concentration of 2%. It is an effective blocker, and will not gel at this concentration at 4°C. Bovine serum albumin (BSA) is available in a wide range of grades. Usually a blotting or immunological grade of BSA is appropriate. It is less expensive than fish skin gelatin, and can be used at 2%. Normal serum (fetal calf or horse) is used sometimes, at a concentration of 10%. It can be an effective blocking agent, but is quite expensive. Since serum contains immunoglobulins, it is not compatible with Protein A and some secondary antibodies. Casein can be used at 1%, but it is very difficult to get dry casein into solution. Casein and casein hydrolysate are the basis of some commercial blocking agents. Different primary antibodies work better with different blocking agents: nonfat dry milk is usually a good first choice, but when setting up a new method, it is a good idea to evaluate different blockers. It has been claimed that some blocking agents, nonfat dry milk in particular, can hide or “mask” certain antigens. Of course, there must be no component of the blocking agent that the primary or secondary antibodies can specifically react with. Some researchers include a second blocking step prior to secondary antibody incubation. However, if the initial blocking is suf- ficient and reagent dilutions are optimal, this should not be necessary. A more specific kind of blocking may be needed when avidin or streptavidin is used as a detection reagent and the sample contains biotin-bearing proteins. Because of this “endogenous biotin” the avidin or streptavidin will pick up these undesired proteins directly. If you suspect this may be a problem, a control reaction can be run with no primary antibody but with the avidin or streptavidin detection. The presence of bands in this control reaction will indicate that the avidin or streptavidin is binding to the endogenous biotin. The remedy for such a situation is to treat the blot prior to antibody incubation first with avidin (to bind all the endogenous Western Blotting 381