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Proteins and Peptides with Capillary Electrophoresis using CElixirPro3

Reference Number: AA-01409 Created: 04/30/2013 02:04 PM Last Updated: 04/30/2013 02:50 PM

Introduction
The CElixirPro3™ kit uses Nitrilotri(trimethyl)phosphonic acid (NTMP) as buffer. NTMP is characterized by 7 pKa’s, allowing the user to design buffers between pH 4 and 7.2. Furthermore it is UV transparent, which makes it about 40 times more sensitive compared to a Tris/taurine or borate buffer. To avoid absorbance of proteins on the capillary, the kit contains double dynamic coating of the capillary (US Patent 5611903).
Methods and Materials
Instrumentation
Suitable instruments are capillary electrophoresis such as PA800 plus or P/ACE MDQ (Beckman Coulter Brea). Typical capillaries have a length of 60.2 cm (50 cm to the detector) and an internal diameter of 50 μm.
Method
The kit contains elements for dynamically coating of the capillary and two separation buffers at pH 4 and 7.2. By mixing both buffers, an intermediate pH may be obtained. The sample may be injected neat or diluted, for example with the sample diluent supplied in the kit.
Separation
Separation of peptides at different pH
Synthetic oligopeptides, used as isoelectric pI markers are mixed and separated at different pH values (Figure 1). At pH 4.4 they all migrate before the electro osmotic peak (EOF), while gradually by increasing the pH they will be negatively charged and migrate after the EOF peak.

Figure 1. Separation of pI markers made of oligopeptides at different pH values.


Separation of Serum Proteins
Serum proteins are composed of a family of proteins and can be separated using agarose gel electrophoresis. Figure 2 shows an Immuno Fixation Electrophoresis, where an IgM kappa monoclonal protein is identified. Lane 1 represents the separation of the serum proteins, while lane 2–6 apply to anti IgG, IgA, IgM, kappa and lambda respectively. Figure 3 shows the same sample analyzed with CElixirPro3™ and immune subtraction to identify the monoclonal band.

Figure 2. Typical separation of serum proteins by agarose gel electrophoresis. The sample contains an IgM kappa monoclonal band.


Figure 3. Same serum protein as in Figure 2, with overlay of immunosubtractions. The electropherogram represents the gamma zone.
Blood products
Intravenous immunoglobulin are prepared from pooled blood and may be used as antibody replacement therapy. It is important to quantify the remaining albumin fraction. Figure 4 show the separation of albumin in presence of immunoglobulin. Short‐end injection is used to increase throughput. Very good linearity is obtained between 0.5–10 mg/mL albumin in the presence of 100 mg/mL IgG.


Figure 4. Overlay of blood immunoglobulin with increased amount of albumin.

Determination of transferrin
During the development of the process of purification of transferrin from blood, the CElixirPro3™ kit is used to quantify the amount of transferrin.

Figure 5. Separation of Apo‐Transferrin at increasing concentration.
Recombinant protein
The stability of a recombinant protein was followed using the CElixirPro3™ kit. Samples were stored at 40°C, 25°C and 5°C and analyzed.

Figure 6. Analysis of recombinant protein stored at different temperatures.
Summary
The CElixirPro3™ kit allows easy analysis by capillary electrophoresis of different proteins and peptides. Good sensitivity and reproducibility are observed for a range of applications.
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