Electrophoresis

SDS-PAGE

SDS-PAGE is a denaturing gel electrophoresis technique used in the development and characterization of protein based products. Sodium dodecyl sulfate (SDS) is a detergent that dissociates and unfolds oligomeric proteins into its subunits. The SDS binds to the polypeptides to form complexes with fairly constant charge to mass ratios. The electrophoretic migration rate through a gel is therefore determined only by the size of the complexes. Molecular weights are determined by simultaneously running marker proteins of known molecular weight.
Samples are typically treated with sample buffer containing SDS at higher temperatures (<90 deg C) in the presence or absence of reducing agents (depending on type of analysis required). Samples are loaded on to either a gradient 4-20% Tris-Glycine gel or normal 10% Tris-Glycine gel (depending on the size of the protein). Mark12 molecular weight marker is used to compare and identify the molecular weight of sample bands. The gel is stained by silver staining using either SilverXpress or SilverQuest silver staining kits or Colloidal blue using Colloidal blue kit. The gels are scanned and analyzed using a densitometer to identify the sample bands.

• Sample required: 100 µg -500 µg protein

CE-IEF

Isoelectric Focusing (IEF) is a characteristics method for the identification and differentiation of proteins. It separates the protein according to their isoelectric points (pI). Proteins are amphoteric molecules; they carry either positive, negative or zero net charge, depending on the pH of their surrounding. When a mixture of proteins is applied at a point in a pH gradient, the different proteins have a different net charge at this pH value. The positively charged proteins migrate towards the cathode, the negatively charged towards the anode, until they reach the pH value, where they are isoelectric. In cIEF technique, protein samples are introduced into a capillary with a mixture of ampholyte buffers which are a polymerized mixture of monomers that contain weakly acidic and weakly basic groups. By using an acidic solution on the anode side of the capillary and a basic solution on the cathode side and applying electric field, the ampholytes molecules orient themselves with respect to their pKa value, generating a pH gradient within the capillary. Proteins migrate through the ampholyte mixture and focus at a pH where they become electrically neutral (pI). After focusing, the zones must be mobilized to the detection window by applying pressure together with focusing potential.
Resolution of pI species on samples is performed by Capillary Isoelectric focusing (cIEF) using a ProteomeLab PA 800 Capillary electrophoresis system. An applicable pI range of 3-10 will be used. Separation is performed on a neutral capillary filled with Ampholyte/sample mixture with a 15 kV focusing voltage applied over 6 minutes. The sample is then mobilized to the detection window at 21kV and 0.5 psi presure. The sample (main peak) and impurities (if any) are detected at 280 nm. The migration time of the sample is correlated with the calibration plot of pI vs the migration time of the pI markers.

• Sample required: 100 µg -500 µg protein

IEF - GEL

Isoelectric Focusing (IEF) is a characteristic method for the identification and differentiation of proteins. It separates the protein according to their isoelectric points (pI). Proteins are amphoteric molecules; they carry either positive, negative or zero net charge, depending on the pH of their surrounding. When a mixture of proteins is applied at a point in a pH gradient, the different proteins have a different net charge at this pH value. The positively charged proteins migrate towards the cathode, the negatively charged towards the anode, until they reach the pH value, where they are isoelectric.
Samples are loaded at different dilutions (1:1/1:10) on to a Serva Precotes pH 3-10 gel along with IEF markers pH 3-10. These are then subjected to electrophoresis using a Multiphor II system.. The gel is stained with either Serva Violet or Colloidal blue, destained and scanned using a densitometer (Molecular dynamics or BIO-RAD GS-800).

• Sample required: 100 µg-500 µg protein

SDS-CE

Capillary methods to determine molecular weight are performed with non-reducing CE-SDS using a ProteomeLab PA 800 Capillary electrophoresis system. Separation is performed on a bare fused silica capillary with -15 kV separation voltage applied over 35 minutes. Intact antibody (main peak) and impurities (if any) can be detected at 220 nm. The migration time / purity of the sample is correlated with designated assay control.

Western Blot

Blotting is the transfer of large molecules on to the surface of an immobilizing membrane. The western blot (protein immunoblot) is an analytical technique used to detect specific proteins in a given sample (i.e. drug product). This blotting technique is used to establish protein identity and purity in respect to host cell protein. Native or denatured proteins are separated by gel electrophoresis (Novex X Cell II system). The proteins are then transferred to a membrane (typically nitrocellulose or PVDF), where they are probed (identified) using antibodies specific to the target protein. After the proteins are transferred to the membrane, the membrane is incubated in series of steps - Incubation with a blocking agent to bind non-specific protein binding sites; Incubation with an antibody with specificity to bind to the protein; Incubation with an alkaline phosphatase secondary IgG to bind with the specific antibody; Incubation with a substrate for alkaline phosphatase to develop color at the site of the protein-antibody complex on the membrane.

• Sample required: 100µg-500µg protein