Colored pI standards and gel isoelectric focusing in strongly acidic pH

Št́astná, Miroslava; Šlais, Karel

doi:10.1007/s00216-005-3106-7

Cited by 36 publications

(33 citation statements)

References 22 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…The examples of focusing patterns of pI markers of various colors can be found in Ref. [11] or at www.iach.cz/liq/projects.html. The steps described in Fig.…”

Section: Resultsmentioning

confidence: 99%

See 1 more Smart Citation

Two-dimensional gel isoelectric focusing

Št́astná

Šlais

2005

Electrophoresis

Self Cite

View full text Add to dashboard Cite

Two-dimensional gel isoelectric focusing (2-D gel IEF) is presented as the combination of the same separation method used consecutively in two directions of the same gel. In this new method, after completion of IEF process in the first dimension the gel was cut into the separate strips, each containing selected analytes together with the appropriate part of the original broad pH gradient, and the strips were rotated by 90 degrees (with regard to the first IEF) and left to diffuse overnight. After diffusion the strips were subjected to the second IEF. During the second IEF, the corresponding narrow part of pH gradient in each strip was restored again, however, now along the strip. The progress of the separation process can be monitored visually by using colored low-molecular-weight isoelectric point (pI) markers loaded into the gel simultaneously with proteins. The unique properties of IEF, focusing and resolution power were enhanced by using the same technique twice. Two forms of beta-lactoglobulin (pI values 5.14 and 5.31, respectively) non-separated in the first IEF were successfully separated in the second dimension at relatively low voltage (330 V) with the resolution power comparable to the high-resolution gels requiring the high voltage during the run and long separation time. Glucose oxidase loaded as diluted solution into ten positions across the gel was finally focused into a single band during 2-D gel IEF. Since the first and second IEF are carried out on the same gel, no losses and contamination of analyte occur. The suggested method can be used for separation/fractionation of complex biological mixtures, similarly as other multidimensional separation techniques applied in proteomics, and can be followed by further processing, e.g., mass spectrometry analysis. The focusing properties of IEF could be useful especially in separation of mixtures, where components are at low concentration levels.

show abstract

“…The examples of focusing patterns of pI markers of various colors can be found in Ref. [11] or at www.iach.cz/liq/projects.html. The steps described in Fig.…”

Section: Resultsmentioning

confidence: 99%

“…The compounds newly synthesized in our group were used as colored pI markers for the indication of the local pH of the pH gradient. They were either azo dyes whose synthesis was described in detail elsewhere [10][11] or they were prepared by the similar procedure as aminomethylated sulfonephthaleins [9] and nitrophenols [8], respectively.…”

Section: Chemicalsmentioning

confidence: 99%

Two-dimensional gel isoelectric focusing

Št́astná

Šlais

2005

Electrophoresis

Self Cite

View full text Add to dashboard Cite

show abstract

“…Further, the CF IEF does not allow the focusing to reach the steady state, making possible the use of simple buffers as carriers instead of the expensive commercial polyampholytes. From many possible carrier electrolytes useful for generation of pH gradient, we prefer here our mixtures of simple defined buffers similar to those described previously [18][19][20], which are based on previous suggestions [3,16,[21][22][23]. The previous concepts were extended here to cover the broad pH range from 3 to 11, the carrier mixture used here contains 23 simple buffers.…”

Section: Solutions and Process Inspectionmentioning

confidence: 99%

Divergent flow isoelectric focusing

Šlais

2008

Electrophoresis

View full text Add to dashboard Cite

Continuous-flow isoelectric focusing (IEF) has the potential to be an important method in proteome analysis. The current devices do not fully use the advantages of IEF, because they do not utilize all its important features including changes in background conductivity during the focusing. A novel continuous-flow IEF method has been developed based on planar divergent flow and control of local electric field by conductivity of electrode electrolytes. A hydrophilized polypropylene nonwoven fabric was used for creation of flow and electric manifold, making the assembled device cheap, flexible and easy to set up and operate. By using the colored low-molecular-weight pI markers we demonstrated much higher speed of focusing in the new designed channel in comparison with a channel based on currently used rectangular geometry. The developed divergent-flow IEF combines the speed of micro flow channels with the separation efficiency and sample load capacity of preparative devices.

show abstract

“…The specifications [41,42] of the used spacers and simple ampholytes have been described in Ref. [43]. The UV detectable pI markers, pI 5 2.0 ( [43] …”

Section: Chemicalsmentioning

confidence: 99%

“…The CIEF experiments were carried out using the laboratory-made apparatus [43] at constant voltage of (À) 20 kV on the side of the detector, supplied by high voltage unit Spellman CZE 1000 R (Plainview, NY, USA). The lengths of the fused-silica capillaries (FS), 0.1 mm id and 0.35 mm od (Pliva-Lachema, Brno, Czech Republic) were 300 and 150 mm to the detector with effective column volumes about 1.2 mL.…”

Section: Equipment and Proceduresmentioning

confidence: 99%

Testing of the influenza virus purification by CIEF

et al. 2010

View full text Add to dashboard Cite

In virological practice, the pre-concentration, purification and subsequent determination of the purity and concentration of the viruses from the cultural medium and/or from the real sample are required. The conventional techniques used today are equipment demanding, time-consuming and laborious. In this study, the CIEF of influenza viruses with UV detection has been developed and subsequently used to test the purification of the virus from the biological samples. The equine and swine influenza viruses present in infected allantoic fluid of specific pathogen free embryonated chicken eggs were precipitated by using PEG 6000 and sodium chloride. The precipitated viruses were centrifuged at 14 000 x g, and the impurities of different densities were removed by using the sucrose gradients. The efficiency of the virus purification technique was examined by the CIEF and compared to the results of real-time PCR. The pIs of both influenza viruses were determined. Simultaneously, the CIEF was found to be a suitable method for the rapid testing of the efficiency of the virus purification.

show abstract

Colored pI standards and gel isoelectric focusing in strongly acidic pH

Cited by 36 publications

References 22 publications

Two-dimensional gel isoelectric focusing

Two-dimensional gel isoelectric focusing

Divergent flow isoelectric focusing

Testing of the influenza virus purification by CIEF

Contact Info

Product

Resources

About