Elimination of basic gaps at high pH values in 2‐DE

Kask, Lena; Larsson, K; Bjellqvist, Bengt

doi:10.1002/pmic.200900091

Cited by 4 publications

(8 citation statements)

References 17 publications

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“…Actually, it offers a good separation of proteins with few horizontal smears [8]. We decided to work with bovine milk proteins, a largely available source of proteins, and with major nutritional interests [10,11].…”

Section: Resultsmentioning

confidence: 99%

“…Protein sample was loaded from wick into the strip during 16 hours at 200 V according to [8]; anodic wick was then removed and replaced with a paper wick soaked with water. Proteins were then focused until 60 KVh-1.…”

Section: Resultsmentioning

confidence: 99%

“…To improve protein focalisation following paper-bridge loading, we tried to remove paper wick bonds between strip and electrode during protein focalisation [8]. With this strategy (Figure 5D), the intensity of some spots around 30 kDa decreased slightly, but a lot of new spots appeared after pH9, and a clear background was observed at the bottom of the gel.…”

Section: Resultsmentioning

confidence: 99%

“…Another possibility consisted to use the “cup loading protocol” described earlier, but instead of using a cup to load proteins, proteins were loaded using the anodic paper wick as described by Kane et al [8], with some adaptations. In this case, strips were first rehydrated using 320 μl of RB with 1.2% destreak reagent and 0.5% IPG buffer 6–11 at 20° for 16 hours.…”

Section: Methodsmentioning

confidence: 99%

“…The use of destreak reagent was proposed to replace DTT to limit unspecific oxidation of protein thiol groups, a combination of isopropanol and glycerol in rehydratation buffer was reported to improve the focusing of proteins in the alkaline IPG strips [6,7]. The paper-bridge loading protocol allowed an improvement of resolution for preparative loading of cardiac mitochondrial samples in the 6–11 pI range [8]. …”

Section: Introductionmentioning

confidence: 99%

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Technical updates to basic proteins focalization using IPG strips

Dépagne

Chevalier

2012

Proteome Sci

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BackgroundGel-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.ResultsThree 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.ConclusionAccording 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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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

See 3 more Smart Citations

Technical updates to basic proteins focalization using IPG strips

Dépagne

Chevalier

2012

Proteome Sci

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Improved consistency in 2D gel electrophoresis: Sheep plasma as a test case

Brown

Norris

2017

Electrophoresis

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The cover photo shows two sets of replicate gels run with sheep plasma samples in 2D elec-trophoresis using different methods (a New Zealand pastoral scene is in the background). Gel consistency and resolution can be a problem with plasma samples run on 2D gels and causes difficulty with spot matching and quantitative analysis. After experimenting with a wide range of parameters a new method was developed for sheep plasma involving conditions at the IPG strip electrodes. The method was tested by analysing the standards from two different DIGE experiments, comparing the old and new methods. Regular Issue Part I: Reviews 769 Assessment of disulfide and hinge modifications in monoclonal antibodies B. Moritz and J. O. Stracke 786 Chiral selectors in CE: Recent development and applications (mid-2014 to mid-2016) I. J. Stavrou, E. A. Agathokleous and C. P. Kapnissi-Christodoulou 820 Flavonoids biosynthesis in plants and its further analysis by capillary electrophoresis

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Optimized two‐dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA‐cisplatin‐protein adducts in ovarian cancer cells

et al. 2018

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Intracellular binding of cisplatin to proteins has been associated with acquired resistance to chemotherapy. In our previous study we established an analytical method for the identification of intracellular cisplatin-binding proteins. The method used a fluorescent carboxyfluorescein-diacetate-labeled cisplatin analogue (CFDA-cisplatin), two-dimensional gel electrophoresis (2DE) and mass spectrometry, which allows detecting and identifying intracellular CFDA-cisplatin-containing protein adducts in the acidic pH range (pH 4-7). Based on this analytical method we extended the identification of intracellular cisplatin-protein adducts to the alkaline pH range (pH 6-10) giving chance to discover new important binding partners. 2DE analysis of alkaline proteins is challenging due to the difficult separation of basic proteins during the isoelectric focusing (IEF). The establishment of an optimized IEF protocol for basic proteins enabled us to identify several intracellular CFDA-cisplatin-binding proteins including enzymes of the glucose and serine metabolism like alpha enolase and D-3-phosphoglycerate 1-dehydrogenase.

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Elimination of basic gaps at high pH values in 2‐DE

Cited by 4 publications

References 17 publications

Technical updates to basic proteins focalization using IPG strips

Technical updates to basic proteins focalization using IPG strips

Improved consistency in 2D gel electrophoresis: Sheep plasma as a test case

Optimized two‐dimensional gel electrophoresis in an alkaline pH range improves the identification of intracellular CFDA‐cisplatin‐protein adducts in ovarian cancer cells

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