What is the best method for applying protein samples to strips for isoelectric focussing?
Cup loading and in-gel rehydration are the most commonly used methods of sample application and can be used with Cup Loading Strip Holders (Code no. 80-6459-43). More CyDye™ DIGE Fluor minimal dye-labelled protein enters the strip using the cup-loading method. The use of longer rehydration times (e.g. 22 h) when using in-gel-rehydration can increase the number of spots detected (particularly in the high molecular weight range), although the overall amount of protein in the strip remains unchanged. The cup-loading method is favored but in-gel rehydration is useful for preparative gels where the sample volume may be too large to apply using the cup-loading approach. Other strip holders available (e.g. Regular 24 cm Strip Holders, Code no. 80-6469-88) allow sample loading via gaps/wells at the sides of the strip holder. CyDye DIGE Fluor minimal dye labelled protein loaded using this method results in levels of protein entry intermediate between cup loading and in-gel rehydration.
