KH 06 Flashcards
What are the 3 separation methods?
Centrifugation, electrophoresis, and chromatography
What are the physical and chemical properties relevant for protein purification and analysis?
Mass or size, density, electrical charge, and binding affinity
What is SDS?
The anionic detergent sodium docecyl sulfate.
When was sodium docecyl sulfate (SDS) developed?
In the late 60s. They are still used daily in almost all molecular biology labs.
What does sodium docecyl sulfate do?
SDS denatures proteins by the interaction of its hydrophobic tail with hydrophobic amino acid side chains, disrupting the oil drop structure of proteins. The hydrophobic
tail of SDS binds not only to hydrophobic residues, but also to itself, so it tends to coat the polypeptide chain in a uniform layer of SDS molecules. Because these are all negatively charged, the various parts of the SDS polypeptide chain repel each other and this further disrupts and unfolds the protein. This unfolding and structural disruption not only completely denatures individual
polypeptides, is separates all the chains of multimeric protein into individual denatured
polypeptides.
What is differential centrifugation?
The separation of cellular contents by particle size/mass.
What is an example of a post-translational modification that has an impact on protein mobility during SDS polyacrylamide gel electrophoresis?
Phosphorylation of proteins by protein kinases can in some cases shift the mobility of the protein, and hence the apparent molecular weight. This mobility effect is thought to result from the phosphate group locally interfering with SDS binding.
What is the isoelectric point?
The pH at which the sum of all charges is 0. It depends the on amino acid composition of each protein.
How is the pH gradient established in isoelectric focusing?
It is established using special buffers, ampholytes, that are immobilized in the acrylamide gel.
What is two-dimensional gel electrophoresis?
Isoelectric focusing followed by SDS PAGE.
What is proteomics?
The analysis of biological protein samples by mass spectrometry and bioinformatics (computer analysis of DNA and protein sequences) in order to identify the population of proteins present in any given subcellular organelle.
