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Technical updates to basic proteins focalization using IPG strips
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 loadi...
Autores principales: | , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
BioMed Central
2012
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517320/ https://www.ncbi.nlm.nih.gov/pubmed/22954324 http://dx.doi.org/10.1186/1477-5956-10-54 |
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author | Dépagne, Jordane Chevalier, François |
author_facet | Dépagne, Jordane Chevalier, François |
author_sort | Dépagne, Jordane |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-3517320 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2012 |
publisher | BioMed Central |
record_format | MEDLINE/PubMed |
spelling | pubmed-35173202012-12-08 Technical updates to basic proteins focalization using IPG strips Dépagne, Jordane Chevalier, François Proteome Sci Methodology 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. BioMed Central 2012-09-06 /pmc/articles/PMC3517320/ /pubmed/22954324 http://dx.doi.org/10.1186/1477-5956-10-54 Text en Copyright ©2012 Dépagne and Chevalier; licensee BioMed Central Ltd. http://creativecommons.org/licenses/by/2.0 This is an Open Access article distributed under the terms of the Creative Commons Attribution License ( http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. |
spellingShingle | Methodology Dépagne, Jordane Chevalier, François Technical updates to basic proteins focalization using IPG strips |
title | Technical updates to basic proteins focalization using IPG strips |
title_full | Technical updates to basic proteins focalization using IPG strips |
title_fullStr | Technical updates to basic proteins focalization using IPG strips |
title_full_unstemmed | Technical updates to basic proteins focalization using IPG strips |
title_short | Technical updates to basic proteins focalization using IPG strips |
title_sort | technical updates to basic proteins focalization using ipg strips |
topic | Methodology |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3517320/ https://www.ncbi.nlm.nih.gov/pubmed/22954324 http://dx.doi.org/10.1186/1477-5956-10-54 |
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