Will the Stripping Procedure Affect the Target Protein ..388 Stripping and Reprobing Can the Same Stripping Protocols Be Used for Membranes from different manufacturers? Is It Always Necessary to Strip a Blot before Troubleshooting 389 Setting Up a New Method Bibliography 397 PHYSICAL PROPERTIES OF PROTEINS What Do You know about Your protein? In order to make informed choices among the bewildering range of available transfer and detection methods, it is best to have as clear an idea as possible of your own particular requirements. In large part these choices will depend on the nature of your target protein Even limited knowledge can be used to advantage How abundant is your protein? It isnt necessary to answer the question in rigorously quantitative terms: an educated guess is suf ficient. Are your samples easy to obtain and plentiful, or limited and precious? Is the sample likely to be rich in target protein(e. g, if the protein is overexpressed)or poor in target(perhaps a cytokine)? Obviously low protein concentration or severely limited sample size would require a more sensitive detection method What is the molecular weight of your target protein? Low MW proteins(12kDa or less) are retained less efficiently than higher molecular weight proteins. Membranes with a pore size of 0.1 or 0.2 micron are recommended for transfer of these smaller pro teins, and PVDF will tend to retain more low Mw protein than nitrocellulose. The ultimate lower limit for transfer is somewhere around 5kDa, although this depends largely on the proteins shape and charge The transfer of high molecular weight proteins(more than 100kDa)can benefit from the addition of up to 0. 1% SDs to the transfer buffer(Lissilour and Godinot, 1990). Transfer time can also be increased to ensure efficient transfer of high molecular ight proteins. What Other Physical Properties Make Your Protein unusual? In cases where proteins are highly basic(where the pl of the protein is higher than the pH of the transfer buffer) the protein 374 RiisStripping and Reprobing . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 388 Will the Stripping Procedure Affect the Target Protein? . . . 388 Can the Same Stripping Protocols Be Used for Membranes from Different Manufacturers? . . . . . . . . . . . 389 Is It Always Necessary to Strip a Blot before Reprobing? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Troubleshooting . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 389 Setting Up a New Method . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 396 Bibliography . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 397 PHYSICAL PROPERTIES OF PROTEINS What Do You Know about Your Protein? In order to make informed choices among the bewildering range of available transfer and detection methods, it is best to have as clear an idea as possible of your own particular requirements. In large part these choices will depend on the nature of your target protein. Even limited knowledge can be used to advantage. How abundant is your protein? It isn’t necessary to answer the question in rigorously quantitative terms: an educated guess is suf- ficient.Are your samples easy to obtain and plentiful,or limited and precious? Is the sample likely to be rich in target protein (e.g., if the protein is overexpressed) or poor in target (perhaps a cytokine)? Obviously low protein concentration or severely limited sample size would require a more sensitive detection method. What is the molecular weight of your target protein? Low MW proteins (12kDa or less) are retained less efficiently than higher molecular weight proteins. Membranes with a pore size of 0.1 or 0.2 micron are recommended for transfer of these smaller pro￾teins, and PVDF will tend to retain more low MW protein than nitrocellulose. The ultimate lower limit for transfer is somewhere around 5kDa, although this depends largely on the protein’s shape and charge. The transfer of high molecular weight proteins (more than 100kDa) can benefit from the addition of up to 0.1% SDS to the transfer buffer (Lissilour and Godinot, 1990). Transfer time can also be increased to ensure efficient transfer of high molecular weight proteins. What Other Physical Properties Make Your Protein Unusual? In cases where proteins are highly basic (where the pI of the protein is higher than the pH of the transfer buffer) the protein 374 Riis