• SDS-UREA PAGE

• ACID UREA PAGE

SDS-UREA PAGESDS-urea PAGE is often used for proteins of low molecular weight and membraneproteins.26,27In SDS-PAGE, the migration of low molecular weight proteins maynot be proportional to their molecular weight, as the protein charge propertiesbecome significant relative to the mass. SDS-urea PAGE is suitable for membraneproteins, as they may not be soluble at conditions used in SDS-PAGE.Working ProcedureAll procedures for SDS-urea PAGE are essentially similar to those described forSDS-PAGE except the composition of gels (both separating and stacking) and sampleloading buffer. These should contain 8 M urea. The recipe for 10% separating gelis shown below as an example

• SDS-UREA PAGE

• ACID UREA PAGE

ACID UREA PAGEPanyim and Chalkley32introduced a continuous acetic acid urea PAGE for theseparation of unmodified histone from its monoacetylated or monophosphorylatedform. The procedure can separate similar basic proteins based on differences ineffective charge as well as differences in size. Proteins of a slightly different chargesuch as unmodified and acidic acetylated derivative can be separated in acid ureaPAGE. Urea is commonly used in this PAGE, mainly to disrupt any aggregation andto increase the density of the loading solution. In this system, riboflavin or riboflavin5′-phosphate is used as initiator of photo-polymerization of acrylamide, as ammon-ium persulfate interferes with stacking of the proteins in the gel. Chloride ions also interfere with the stacking system. Thus, protein samples and glycine used in theelctrophoresis buffer should be free of chloride saltsMechanismIn acid urea PAGE, the samples are electrophoresed in acetic acid buffer (pH around3). At this pH, basic proteins with a high isoelectric point get positively charged andmove toward the cathode under an electric field.3

Procedure1.Prepare gel sandwich as shown in Figure 3.4 A.2.In a 100 ml side-arm flask, combine the following reagents for separatinggel without riboflavin 5′-phosphate and TEMED. The recipe is good formaking five mini gels.