In the second dimension, polypeptides are separated according to their size in a gel matrix. Practically, after electric focusing in reducing conditions, proteins are separated into their polypeptide components. The latter are mixed with SDS-containing buffers. SDS binds to polypeptides at a constant mass ratio (1.4 g SDS per gram of protein). As a consequence, poly-peptides organize as rod-like molecules, with a diameter of 1.8 nm, and their lengths depend on their molecular weight. The bound SDS molecules contribute to a strong negative charge, which effectively swamps the intrinsic charge of the polypeptides. Thus, in general the SDS-polypeptide complexes have the same mass/charge ratio and, in a sieving polyacrylamide matrix, they will migrate according to their molecular weight. Glycoproteins and lipop-roteins can migrate abnormally as they are not easily saturated with SDS.

The gel matrix is most frequently composed of polycrylamide generated by polymerization of acryl-amide monomers into long chains that are cross-linked. Usually, cross-linkers are bifunctional components such as N,N'-methylenebisacrylamide (bis) or diacrylpiperazine. Polymerization of acrylamide is initiated either by the use of ammonium persulfate or riboflavin, and is accelerated by the use of N,N,N',N'-tetramethylethylenediamine (TEMED). Oxygen inhibits polymerization, and thus gel mixtures are usually degassed. The composition of a polyacrylamide gel is defined by two parameters: % T and % C. The % T (w/v) is the total concentration of the monomer (acrylamide plus cross-linker), whereas % C corresponds to the ratio (w/w) of the cross-linker to the acrylamide. The pore size of the polymerized acryl-amide will depend on these two parameters, but since pore formation is random, pore sizes will never be totally uniform. The choice of the mean pore size will depend on the molecular weight of the proteins to be studied. The second-dimension SDS-PAGE can be performed with home-made vertical or horizontal systems, using linear or gradient polyacrylamide

(9-16%) gels. Commercially manufactured gels are also available. The advantages of the latter reside in their reproducibility and safety (polymerized acrylamide being clearly less neurotoxic as compared with monomeric acrylamide). The sensitivity as well as the resolution power of the protein detection must be kept in mind before choosing optimal conditions for SDS-PAGE. Gels polymerized with the photoinitiator system, composed of methylene blue, toluene sulflnate and diphenyliodonium chloride, lead to low resolution power after silver staining. Resolution can be restored if methylene blue is replaced by riboflavin. Gels polymerized with the riboflavin/sulfinate/iodonium system yield better results upon N-terminal microsequencing after blotting than gels polymerized with the standard TEMED/ammonium persulfate system.

Solar Panel Basics

Solar Panel Basics

Global warming is a huge problem which will significantly affect every country in the world. Many people all over the world are trying to do whatever they can to help combat the effects of global warming. One of the ways that people can fight global warming is to reduce their dependence on non-renewable energy sources like oil and petroleum based products.

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