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.) RiisPVDF (polyvinylidene difluoride) membranes are physically stronger and have higher protein-binding capacity than nitrocel￾lulose. 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