When hydrophobic samples, or membrane proteins, are disaggregated in buffers containing detergents (e.g. Nonidet P-40), urea and 2-mercaptoethanol, and applied at the cathodic end of a gel cylinder or slab for isoelectric separation, as routinely performed for two-dimensional techniques, a severe disturbance of the alkaline region of the pH gradient ensues. This phenomenon has been attributed to high protein loads, which supposedly overcome the buffering power of isoelectric carrier ampholytes. On the contrary, in the present study it has been found that this suppression of the alkaline end of the pH gradient is due to 2-mercaptoethanol, which is a buffer with pK 9.5. This compound ionizes at the basic gel end and is driven electrophoretically along the pH gradient, sweeping away, along its path, the focused carrier ampholytes.

Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing / P.G. Righetti, G. Tudor, E. Gianazza. - In: JOURNAL OF BIOCHEMICAL AND BIOPHYSICAL METHODS. - ISSN 0165-022X. - 6:3(1982 Aug), pp. 219-227. [10.1016/0165-022X(82)90044-6]

Effect of 2-mercaptoethanol on pH gradients in isoelectric focusing

E. Gianazza
Ultimo
1982

Abstract

When hydrophobic samples, or membrane proteins, are disaggregated in buffers containing detergents (e.g. Nonidet P-40), urea and 2-mercaptoethanol, and applied at the cathodic end of a gel cylinder or slab for isoelectric separation, as routinely performed for two-dimensional techniques, a severe disturbance of the alkaline region of the pH gradient ensues. This phenomenon has been attributed to high protein loads, which supposedly overcome the buffering power of isoelectric carrier ampholytes. On the contrary, in the present study it has been found that this suppression of the alkaline end of the pH gradient is due to 2-mercaptoethanol, which is a buffer with pK 9.5. This compound ionizes at the basic gel end and is driven electrophoretically along the pH gradient, sweeping away, along its path, the focused carrier ampholytes.
2-mercapthoethanol; isoelectric focusing; two-dimensional techniques
Settore BIO/10 - Biochimica
ago-1982
Article (author)
File in questo prodotto:
Non ci sono file associati a questo prodotto.
Pubblicazioni consigliate

I documenti in IRIS sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.

Utilizza questo identificativo per citare o creare un link a questo documento: https://hdl.handle.net/2434/176491
Citazioni
  • ???jsp.display-item.citation.pmc??? 2
  • Scopus 54
  • ???jsp.display-item.citation.isi??? 57
social impact