basicpeptides

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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		IEF of basic peptides


	The more basic the gradient the unsharper the bands (Peter Reiter, Gelita, Eberbach, Germany)


	SDS of basic proteins and peptides



		Basic proteins and peptides in SDS. Not good! (Peter Reiter, Gelita, Eberbach, Germany)


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Basic Peptides


	Basic peptides don’t like to be driven to their isoelectric points! What they like is to have their maximal positive charges. They also have no affinity to the negatively charged SDS-molecule. So they should be separated in a native cathodal electrophoresis. An acidic buffer will do this in a perfect way.


	SDS of basic peptides


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		Basic peptides don’t like SDS (Peter Reiter, Gelita, Eberbach, Germany)




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		Native cathodal electrophoresis: Basic peptides want to be separated in an acidic buffer. Here they are charged (+)!



	Acidic buffer = cathodal electrophoresis (Peter Reiter, Gelita, Eberbach, Germany)


			5-7 kD

		11 kD

				3 kD

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	In native electrophoresis you will have no relation to mol-weight! Double dimensional electrophoresis shows this. First dimension = native cathodal electrophoresis Second dimension = SDS-electrophoresis


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	<------------- cathodal native electrophoresis


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				April 28, 2023

		Native cathodal electrophoresis: Basic peptides / proteines of E.coli



			Acidic buffer = cathodal electrophoresis


	SDS-electrophoresis

		100 kD

				I I I I I I I I I I I

			50 kD

					I I I I I I I I I I I I I I I I

			10 kD


	If SDS shows no good results as second dimension: Double Native Electrophoresis First dimension = IPG-strip pH 7-11, 11 cm Second dimension = cathodal native electrophoresis Visualization: Hot Coomassie or Semi-Colloidal Coomassie


	pH 7


	<------------- IPG-strip 7-11 ----------->


	pH 11


	Basic peptides / proteins of E.coli in double native electrophoresis Molweights are not relative to their migration speeds.


	Native cathodal electrophoresis

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	EDC Electrophoresis Development & Consulting, Vor dem Kreuzberg 17, 72070 Tübingen (Germany)


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