Technical updates to basic proteins focalization using IPG strips.
Proteome Sci
; 10(1): 54, 2012 Sep 06.
Article
em En
| MEDLINE
| ID: mdl-22954324
ABSTRACT
BACKGROUND:
Gel-based proteomic is a popular and versatile method of global protein separation and quantification. However, separation of basic protein still represents technical challenges with recurrent problems of resolution and reproducibility.RESULTS:
Three different protocols of protein loading were compared using MCF7 cells proteins. In-gel rehydration, cup-loading and paper-bridge loading were first compared using 6-11 IPG strips, as attempted, in-gel rehydration gave large horizontal steaking; paper-bridge loading displayed an interesting spot resolution, but with a predominant loss of material; cup-loading was selected as the most relevant method, but still needing improvement. Twelve cup-loading protocols were compared with various strip rehydration, and cathodic wick solutions. Destreak appeared as better than DTT for strip rehydration; the use of isopropanol gave no improvement. The best 2DE separation was observed with cathodic wicks filled with rehydration solution complemented with DTT. Paper-bridge loading was finally analyzed using non-limited samples, such as bovine milk. In this case, new spots of basic milk proteins were observed, with or without paper wicks.CONCLUSION:
According to this technical study of basic protein focalization with IPG strips, the cup-loading protocol clearly displayed the best resolution and reproducibility strips were first rehydrated with standard solution, then proteins were cup-loaded with destreak reagent, and focalisation was performed with cathodic wicks filled with rehydration solution and DTT. Paper-bridge loading could be as well used, but preferentially with non-limited samples.
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Base de dados:
MEDLINE
Idioma:
En
Revista:
Proteome Sci
Ano de publicação:
2012
Tipo de documento:
Article
País de afiliação:
França