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Immunoblotting is typically used to determine the amount (dot blot) and molecular weight (western blot) of an antigen present in a complex mixture. Assay sensitivity can be modulated by using different enzyme substrates and/or by using biotin-(strept)avidin conjugates in place of an enzyme conjugated secondary antibody. The highly sensitive procedure below is suggested. If decreased sensitivity is desired, replace the biotin-streptavidin conjugate with an antibody-peroxidase conjugate. All incubations are at room temperature.
This procedure is for nitrocellulose or PVDF membranes. Do not to touch the membrane! Wear gloves. Nylon membranes may be used, however, they are more difficult to block. To block nylon membranes use buffer without Tween-20, replace BSA with 10% nonfat dry milk and block for several hours to overnight at 37°C.
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