A wide range of analytical techniques can be used to measure the various properties of a protein, such as the size, charge, amino acid composition and hydrophobicity. Confirmation of the purity of growth factors by several varied techniques ensures reliability of results. When shopping for growth factors look for products where purity has been validated by multiple methods. Some common purity assessment methods are discussed below.
What is an SDS-PAGE gel and how do you interpret it? What are reducing vs non-reducing conditions?
SDS-PAGE (sodium dodecyl sulfate – polyacrylamide gel electrophoresis) is a method that separates protein by its mass. SDS is an ionic detergent that denatures and binds to proteins to make them uniformly negatively charged. This means that when an electronic current is applied to a polyacrylamide gel, the SDS bound proteins will migrate down the gel towards the positively charged electrode, separated by size alone.
Proteins can be reduced before being run on an SDS-PAGE gel. In reducing conditions β-mercaptoethanol (β-ME or 2-ME) or dithiothreitol (DTT) is added; this reduces the disulphide bridges in proteins so that when they are run on the gel, they are better separated by size. Many of our growth factors are disulfide-linked dimers and so running non-reduced proteins on the same gel as reduced proteins allows confirmation of the correct dimeric state.
What should I look for from mass spectrometry analysis?
Mass spectrometry analysis is used to confirm the molecular mass of the intact protein and to reveal any heterogeneity that would not be evident from SDS-PAGE analysis. The resultant mass is compared with calculated mass of the protein with the assumption that all the cysteines are disulfide-linked. Multiple peaks represent different charge states of the protein.
What does analytical reversed-phase chromatography show?
Analytical reversed-phase chromatography is used to assess protein purity and structural homogeneity. Homogeneity is judged by the absence of multiple peaks and by the symmetry of the main peak. In these traces look for a single sharp peak; this indicates a single species is present in the sample with no contamination.
Endotoxin levels; what problems can endotoxins cause? What level of endotoxin contamination is acceptable?
Endotoxins are small hydrophobic lipopolysaccharide molecules, a toxic substance found in the outer cell membrane of gram-negative bacteria. Endotoxins are shed by bacteria during their cell death or when they are actively growing and dividing. Endotoxins can affect the growth or performance of cell cultures and are a major source of experimental variation. Endotoxins have a very high heat stability meaning they cannot be destroyed with regular decontamination methods such as autoclaving. Endotoxins are also hydrophobic, and consequently have a strong affinity for other hydrophobic materials such as lab plastics. Endotoxins can be avoided by cleanliness and proper lab techniques to keep endotoxin levels in a lab at bay. At Qkine, we achieve this with good aseptic technique, filter sterilization of buffers used during manufacturing processes and rigorous purification column cleaning protocols. This will minimize introduction of endotoxins into any manufacturing/research processes.
To determine the presence of endotoxins, a limulus amebocyte lysate (LAL) assay is used. This assay can detect as little as 0.01 endotoxin units (EU)/mL. One EU equals approximately 0.1 to 0.2 ng endotoxin/mL of solution. Current industry standards require endotoxin levels in growth factor lots to be less than 0.5 EU/mL, however the lower the better, and companies such as Qkine impose internal standards of <0.1 EU per µg protein.