EDC Impressum & DSE

Electrophoresis
Development & Consulting
Dr. Hanspeter Schickle
Wolfgang Gstrein

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Native Electrophoresis

Mai 30, 2018

Proteins:
1.
If different proteins with the same molecular size should be separated, or if the proteins should still function after electrophoresis, there are 2 possibilities to do that:
2. If Small proteins or peptides should be separated and in SDS they run to quick (with the front):
The Isoelectric Focusing (IEF) or Native, anodal/cathodal Electrophoresis
There are several advatages for the use of native proteins instead of the IEF:
1. Quicker  2. Cheeper  3.  Less complicated staining methods (to be stained like SDS-gels)
To decide if an anodal electrophoresis (anions in a basic buffer system) or a cathodal electrophoresis (cations in an acidic buffer system) should be used, the isoelectric points (IP) of the proteins should be known. In general the acidic proteins (IP below ph 7) should run in a basic pH towards the anode as anions, and the basic proteins (IP over pH 7) should run in a acidic pH towards the cathode as cations.
Native electrophoresis will not deliver a pysical value like pI or MW. Native bands should be type in dfferent ways: Comparing to bands of known proteins.
Pattern in native electrophoresis are compared to other (known) patterns to determine the proteins.
Or different patterns are compared to each other wether there are differences or not.
The markers are used only to differ between problems in electrophoresis and problems with the samples themselves!

Native electrophoresis lanes can serve as a first dimension. This combination is called
 “Double Dimensional Electrophoresis”. Then SDS is the second dimension.
Native electrophoresis can serve as a second dimensions:
“Double Native Electrophoresis”. Then IEF serves as first dimension.

DNA:
Run only anodal as double stranded or single stranded samples or on denaturing gel.

Double Stranded DNA

Double Stranded DNA

[anodal]
[cathodal]
[Double Native]
[Double-Dimensional]
[Native Trouble]

DNA, proteins, peptides

proteins, peptides

proteins, peptides

proteins

Single Stranded DNA

Single Stranded DNA

Protein

Your target molecules -- which method?

Molecule

Method

DNA, RNA

Run only anodal

Acidic Protein

Anodal (basic buffer)

Neutral Protein

Anodal or cathodal

Basic Protein

Cathodal (acidic buffer)

Interpretation of results

The electrophoretic equation is valid: V=Q/M. The proteins do not run according to their molweight. They are driven by charge and hindered by molweight

DoubleNativeFalschfarbenKlein