Isoelectric Focusing

In 1964, synthetic carrier ampholytes (aliphatic oligoamino and oligocarboxylic acids) were synthesized allowing separation of peptides according to their charges. For 2D-PAGE, carrier ampholyte isoelectric focusing is usually performed in an am-pholyte-polyacrylamide matrix that is polymerized in a glass tube. After sample loading, polypeptides are concentrated into narrow bands within a continuous pH gradient in the polyacrylamide gel matrix. Proteins migrate in an electric field until they arrive at a position in which they have no net charge, i.e. their isoelectric point (pi). Isoelectric focusing is useful because: (i) no diffusion of proteins occurs because of the focusing effect; (ii) it offers a resolution allowing the separation of microheterogeneous populations of proteins; and (iii) the pi of the protein can be estimated. In the past, pH gradients were generated by carrier ampholytes or amphoteric buffer moving freely within an acrylamide matrix.

Practically, many factors can affect measurement of the apparent pi of the proteins during isoelectric focusing with carrier ampholytes: (i) since the proteins have their own inherent charges, they can act as ampholytes themselves and affect pH during focus ing; (ii) the carrier ampholytes have a higher mobility than the proteins; (iii) some proteins may never reach steady state due to polyacrylamide gel matrix restriction; (iv) ampholyte-protein interactions may alter the observed pi of the proteins. Temperature, time, voltage and salt concentration are other parameters that may dramatically influence the determination of the pi. Finally, the basic proteins are not detected without using nonequilibrium pH gradient elec-trophoresis. Nowadays, many of these problems have been resolved by the development of isoelectric focusing with an IPG. The pH gradient is created by copolymerization of acrylamide/N,N'-methylene-bisacrylamide with acrylamido derivatives, containing either carboxyl or tertiary amino groups as buffers and sulfate groups (acidic) or quaternary ammonium (basic) as strong titrants (Immobilines). This method is a true equilibrium method, which significantly improves the feasibility of the 2D-PAGE.

Recently, highly reproducible, commercially available, wide-range as well as narrow-range IPGs have been produced. The latter gradients allow a pH scale that enables comparison of several 2D gel maps generated with many IPGs in the first dimension and with various biological samples. IPGs also offer the possibility of determining pi without major differences from the calculated pi values, unless there are significant post-translational protein modifications. Nowadays, with the improvements of IPG production, it is possible to detect proteins with pis up to pH 12 in a single IPG gel with highly reproducible protein patterns.

Finally, as mentioned in the previous section, entire IPG gels can be used for sample application, with the protein entering the gels during their rehydration. This approach is useful because it eliminates precipitation at the sample application site, it improves the resolution over the entire pH range of the gels, and it allows precise control of protein amounts and sample volumes loaded on to the IPG gels. Up to 5 mg of proteins can be loaded on wide IPG gels and up to 15 mg on narrow pH range gels. Contrarily to iso-electric focusing with carrier ampholytes, electroen-dosmosis (transport of water towards the cathode at low pH values or towards the anode at high pH values) is generally not a problem with IPGs. Currently, isoelectric focusing using carrier ampholytes still has a place in a 2D laboratory, because the resolution of particular proteins is sometimes better.

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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