Preparative divergent flow IEF without carrier ampholytes for separation of complex biological samples.
Efficient separation method is a crucial part of the process in which components of highly complex biological sample are identified and characterized. Based on the principles of recently newly established electrophoretic method called divergent flow IEF (DF IEF), we have tested the DF IEF instrument which is able to operate without the use of background carrier ampholytes. We have verified that during separation and focusing of sample consisting of high numbers of proteins (yeast lysate and wheat flour extract), the pH gradient of preparative DF IEF can be created by autofocusing of the sample components themselves without any addition of carrier ampholytes. In DF IEF, the proteins are separated, desalted and concentrated in one step. The fractions of yeast lysate sample, collected at the DF IEF output and subjected to gel IEF, contained the zones of proteins gradually covering the pI values from 3.7 to 8.5. In our experimental arrangement, the highest number of proteins has been found in fractions with pI values around 5.3 as detected by polyacrylamide gel IEF with CBB staining. During DF IEF, the selected protein bands have been concentrated up to 16.8-fold.
Stastna
Miroslava
M
Institute of Analytical Chemistry of the ASCR, Brno, Czech Republic. stastna@iach.cz
Slais
Karel
K
D000665:Ampholyte Mixtures; D053000:Analytic Sample Preparation Methods / Q000295:instrumentation / Q000379:methods*; D001688:Biological Products / Q000737:chemistry / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007525:Isoelectric Focusing / Q000295:instrumentation / Q000379:methods*; D010936:Plant Extracts / Q000737:chemistry / Q000302:isolation & purification; D011506:Proteins / Q000737:chemistry / Q000302:isolation & purification; D014908:Triticum / Q000737:chemistry; D015003:Yeasts / Q000737:chemistry
D000665:Ampholyte Mixtures; D001688:Biological Products; D010936:Plant Extracts; D011506:Proteins