Proteins

Prior to the analysis of proteins many aspects have to be considered. One the one hand the isoelectric point of the proteins should be known to decide whether the proteins are measured as cations or anions. On the other hand the tendency of proteins to interact with the wall of the capillary has to be taken into account, in some cases coated capillaries are preferable. Finally it has to be decided whether the proteins are analysed in their natural folded in their denaturated form. This decision has consequences on the choice of several method parameters like the composition of the electrolyte or the temperature.

Standard proteins

  • Separation: MEKC
  • Electrolyt: Boraet-SDS
  • Capillary: fused silica, 50 µm ID, bubble cell, 64 cm in total
  • Detection: direct UV, 200 nm
  • Description: The standard solution contains 100 mg ribonuclease, lysozyme, cytochrome and myoglobin, each. The interaction of the proteins with the wall of the capillary is suppressed due to the micellar electrolyte.
Proteine_Standard

Protein mixture standard solution

Download: Proteine_Standard.pdf

Proteins in milk (1,5%)

The most important proteins in milk are caseine, β-lactoglobulin and α-lactalbumin. They are analyzed with capillary electrophoresis in several dairy products. Due to the specific composition of the gel even the determination of the individual phosphorylic forms of caseine is possible.

  • Separation: Gel electrophoresis (CGE)
  • Electrolyte: 6 M urea, MHEC, citrate, pH 3
  • Capillary: coated, 50 µm ID, 64 cm in total
  • Detection: direct UV, 210 nm
  • Description: A traditionally produced raw milk with 1,5 % fat was analyzed. The sample was prepared with urea and DTT. The interaction of the proteins with the wall of the capillary was inhibited due to the application of a coated capillary. The high selectivity allows the distinction of the different caseines.
Proteine, milk, Milch, casein, lactoglobulin, lactalbumin

Proteins in milk, 1,5%

Download: Proteine_Milch_1.pdf

Proteins in milk (3,5%)

  • Separation: Gel electrophoresis (CGE)
  • Electrolyte: 6 M urea, MHEC, citrate, pH 3
  • Capillary: coated, 50 µm ID, 64 cm in total
  • Detection: direct UV, 210 nm
  • Description: The pasteurized milk with 3,5 % fat was prepared with urea and DTT. The interaction of the proteins with the wall of the capillary was inhibited due to the application of a coated capillary. The high selectivity allows the distinction of the different caseines.
Proteine_Milch_2

Proteins in milk 3,5%

Download: Proteine_Milch_2.pdf

SDS-MW: Determination of the size of proteins with CE

When using the SDS-MW technique the determination of the size of proteins is possible with CGE due to logarithmical calibration.

  • Separation: Gel electrophoresis (CGE)
  • Electrolyte: Sample suffer: 100 mM Tris-HCl pH 9.0, 1% SDS
  • Gel: SDS-MW Gel Buffer pH 8.0, 0,2% SDS (Sciex, A30341)
  • Capillary: fused silica, 50 µm ID, 33 cm in total, 24 cm effective
  • Detection: direct UV, 200 nm
  • Injection: elektrokinetic, -8 kV, 20 s
  • Description: The given example shows the calibration of a molecular weight standard with the value table and the calibration curve. The molecular weight standard contains seven standard substances from 10 kDa up to 225 kDa. The 10 kD standard is used as internal standard. Due to the high viscosity of the gel it is essentially to inject the sample electrokinetically. In case of high ionic strength in the sample a desalination has to be executed prior to the injection to prevent discrimination of the protein during the injection process.
SDS-MW, CGE

Determination of the size of proteines with SDS-MW-CGE

Download: SDS-MW.pdf

SDS-MW: Dezermination of the size of proteines with CE in a quick screening

  • Separation: Gel electrophoresis (CGE
  • Electrolyte: Sample suffer: 100 mM Tris-HCl pH 9.0, 1% SDS
  • Gel: SDS-MW Gel Buffer pH 8.0, 0,2% SDS (Sciex, A30341)
  • Capillary: fused silica, 50 µm ID, 33 cm in total, 24 cm effective
  • Detection: direct UV, 200 nm
  • Injection: elektrokinetic, -8 kV, 20 s
  • Description: The calibration of a molecular weight can be done as a quick screening when the separation lenght is shortened to 8 cm. The whole calibration is complete in 10 min. The given example shows the calibration of the molecular weight standard with the value table and the calibration curve. The standard contains seven standard substances from 10 kDa up to 225 kDa.
SDS-MW, CGE, CE, Kapillarelektrophorese

Determination of the size of proteines with CE screening

Download: SDS-MW-Screening.pdf