2.7. Membrane Stripping
In principle, “stripping” of a membrane from the interacting protein it has been exposed to should be possible, resulting in regeneration of the membrane for subsequent use. It has been suggested that membranes can be regenerated up to 20 times [28], however if the binding to the SPOTed peptides is very strong, stripping may not yield a re-usable membrane. Several stripping protocols have been reported [10,27,29] employing different chemicals as well as different washing steps. In principle membranes are treated with chemicals aiming to denature the bound protein (e.g., β-mercaptoethanol, urea, guanidinium) together with a detergent (SDS or Triton X-100) which helps lift the protein from the membranes. Affinity chromatography matrixes, such as Ni-NTA agarose beads (Qiagen, Manchester, UK) or Talon Metal Affinity Resin (Clontech, Mountain view CA, USA), may help trapping the unbound protein, thus avoiding re-deposition onto the membrane. Subsequent treatment with TFA (ranging from diluted to highly concentrated) for up to 12 h, has been used to regenerate commercially available membranes (which seem to be quite resistant to acidic pH). Lower acid concentrations are required for in-house functionalized membranes which tend to be more prone to hydrolysis and loss of the ester used to attach the peptides on the sheet support. It is important that membranes remain wet following probing of interactions, in order to perform stripping steps. In addition, regeneration should be monitored by repeating the detection step in the absence of any protein sample in order to ensure that the membranes are clean and ready for re-use. We had mixed results with the regeneration of membrane carrying acetylated peptides, sometimes resulting in incomplete removal of the bound proteins. For this reason we typically used new in-house functionalized membranes for every experiment in order to avoid incomplete stripping.