Native PAGE

Native PAGE

Native polyacrylamide gel electrophoresis is an electrophoretic separation method typically used in proteomics and metallomics.

Native PAGE separations are run in non-denaturing conditions. Detergents are used only to the extent that they are necessary to lyse lipid membranes in the cell. Complexes remain--for the most part--associated and folded as they would be in the cell. One downside, however, is that complexes may not separate cleanly or predictably, since they cannot move through the polyacrylamide gel as quickly as individual, denatured proteins.

Take care not to confuse Native PAGE with SDS-PAGE. SDS-PAGE uses a detergent, sodium dodecyl sulfate, to transfer a negative charge dependent on the mass to the proteins and allow the electrophoretic separation. Due to the repulsion of equal charges, proteins are denatured and protein complexes are separated, reducing the dependence of protein mobility on folding and allowing for a reproducible separation after the mass of the proteins.

There are three popular methods of native PAGE, "clear native" (CN-PAGE), "blue native" (BN-PAGE), and "quantitative preparative native continuous" (QPNC-PAGE).

Blue Native PAGE

BN-PAGE is the oldest native PAGE technique, where the Coomassie blue dye procures the necessary charges to the protein complexes for the electrophoretic separation. The disadvantage of coomassie is that in binding to proteins it can act like a detergent causing complexes to dissociate. Another drawback is the potential quenching of chemoluminescence (e.g. in subsequent western blot detection or activity assays) or fluorescence of proteins with prosthetic groups (e.g. heme or chlorophyll) or labelled with fluorescent dyes.

Clear Native PAGE

CN-PAGE uses no dye, which makes it necessary to use other means to transfer sufficient charge to the proteins, like the use of SLS. CN-PAGE separates acidic water-soluble and membrane proteins (pI < 7) in an acrylamide gradient gel, and usually has lower resolution than BN-PAGE. However, this method is arguably the most useful for examining protein-protein interactions, particularly in conjunction with mass spectrometry (MS).

Quantitative Preparative Native Continuous PAGE

In contrast to CN and BN-PAGE the protein complexes of interest (metalloproteins) may separate cleanly and predictably in fractions, since they move through the polyacrylamide gel as quickly as individual, denatured proteins due to pH 10.00 of the applied electrophoresis buffer and specific properties of the gel column and separation chamber. QPNC-PAGE may be coupled off-line to size exclusion chromatography (SEC), ICP-MS and NMR for structural determination of bioactive and denatured metallobiomolecules in complex protein mixtures. Furthermore, the QPNC-PAGE procedure is a protein conformational-sensitive (PCS) method and in addition, different isoforms of metalloproteins can be isolated in specific PAGE fractions.

Downstream processing

In a typical native PAGE experimental procedure, the complexes will be separated with CN or BN-PAGE. An additional separation method may then be used, such as isoelectric focusing or SDS-PAGE. The gel will then be physically cut, and the protein complexes extracted from each portion separately. Each extract may then be analysed, such as by peptide mass fingerprinting or de novo sequencing after in-gel digestion. This can provide a great deal of information about the identities of the proteins in a complex.

ee also

* SDS-PAGE
* Proteomics
* Mass spectrometry
* QPNC-PAGE

External links

* [http://www3.interscience.wiley.com/cgi-bin/abstract/113343976/ABSTRACT Discontinuous native protein gel electrophoresis]


Wikimedia Foundation. 2010.

Игры ⚽ Нужен реферат?

Look at other dictionaries:

  • Native-PAGE — Nativ Gelelektrophorese (CN PAGE) (engl. colorless native polyacrylamide gel electrophoresis) ist eine Variante der Gelelektrophorese, bei der native, also gefaltete Proteine im elektrischen Feld durch das Molekularsieb eines Polyacrylamid Gels… …   Deutsch Wikipedia

  • Native American disease and epidemics — pervade many aspects of Native American life, both throughout history and in the modern day. Diseases and epidemics can be chronicled from centuries ago when European settlers brought diseases that devastated entire tribes to the modern day when… …   Wikipedia

  • Native Tongue (Suzette Haden Elgin novel) — Native Tongue   Author(s) Suzette Haden Elgin …   Wikipedia

  • Native Guns — Origin Los Angeles, California, US Genres Hip hop Years active 2002 2007 Members Bambu …   Wikipedia

  • Native Gel Electrophoresis — is a technique used mainly in protein electrophoresis where the proteins are not denatured and therefore separated based on their charge to mass ratio.The two main types of native gels used in protein electrophoresis are polyacrlylamide gels and… …   Wikipedia

  • Native American jewelry — is the personal adornment, often in the forms of necklaces, earrings, bracelets, rings, pins, brooches, labrets, and others, made by the Indigenous peoples of the United States. Native American jewelry reflects the cultural diversity and history… …   Wikipedia

  • Native Community Lands — (Spanish: Tierra Comunitaria de Origen, acronym: TCO), according to Bolivian law, are territories held by indigenous people through collective title. The creation of these territories has been a major goal of Bolivian indigenous movements and a… …   Wikipedia

  • Native Oberon — or ETHNO, is the version of the Oberon operating system which runs on x86 PC hardware. It has minimal hardware requirements (133MHz Pentium, 100MB hard disk, and a VESA 2 graphics card with a resolution of at least 1024x768 pixel, optionally a… …   Wikipedia

  • Native Son (disambiguation) — Native Son or Native Sons can be several things: Fiction Native Son is a 1940 novel by Richard Wright. Native Son (1951 film) Native Son (1986 film) History Native Sons of the Golden West, a Californian group Music Native Sons, the sixth album by …   Wikipedia

  • Native Indonesians — Pribumi Total population 230 million (2006 estimation)[1] Reg …   Wikipedia

Share the article and excerpts

Direct link
Do a right-click on the link above
and select “Copy Link”