Gels and Electrophoresis

These separate molecules by size—smaller ones move farther. 

Generating a RECOMBINANT-DNA molecule using restriction enzymes to generate ends that can be joined in a specific fashion. 

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Figure 4.12. Two-Dimensional Gel Electrophoresis. (A) A protein sample is initially fractionated in one dimension by isoelectric focusing as described in Figure 4,11. The isoelectric focusing gel is then attached to an SDS-polyacrylamide gel, and electrophoresis is performed in the second dimension, perpendicular to the original separation. Proteins with the same pi are now separated on the basis of mass. (B) Proteins from E. coli were separated by two-dimensional gel electrophoresis, resolving more than a thousand different proteins. The proteins were first separated according to their isoelectric pH in the horizontal direction and then by their apparent mass in the vertical direction. [(B) Courtesy of Dr. Patrick H. O Farrell.]...

Northern blot technique A technique that is used to identify RNA. RNA is separated according to size by use of a denaturing gel and electrophoresis prior to being blotted onto a solid support. The mRNA transcripts are then detected by hybridization with a radioactive labelled probe. The abundance of the mRNA is indicated by the intensity of the radioactive signal. See also Southern blot technique. NOS nitric oxide synthase, nosocomial synonomous with HAL NO synthase See nitric oxide synthase. 

FIGURE 9.2 The rise and fall of two-dimensional gels. The number of articles published each year according to a Medline search to find TWO and DIMENSIONAL and GEL and ELECTROPHORESIS. 

Correctly dilute the concentrated buffer for preparation of both the gel and electrophoresis (chamber) buffer. Remember that without buffer in the gel, there will be no DNA mobility. Use only distilled water to prepare buffers. Do not use tap water. 

The protein mixture is first separated by IFF (first dimension). This gel is then laid alongside a rectangular SDS slab gel, and electrophoresis run perpendicular to the original direction. Proteins are therefore separated in two dimensions on the basis of both their apparent size (SDS-PAGE) and pi (lEF). Several thousand different proteins can often be resolved in this way. 

A photograph of one such gel, with protein bands made visible by Goomassie Blue staining is shown at the top of the figure ( 6 stains very poorly with this dye). An identical gel was treated with NH OH and GH COOH to cleave the crosslinks, then place on top of an SDS-polyacrylamide slab gel, and electrophoresis in the second dimension was carried out. After the gel was dried, 5h- labeled protein spots were visualized by autoradiography. The arrows indicate the direction of migration of proteins during electrophoresis. 

Minimize the exposure of the DNA to the UV light as best you can. Too long of an exposure can render the DNA unusable. The addition of 1 mM guanosine or cytidine to the gel and electrophoresis buffer is effective in protecting DNA against UV-induced damage. Also, see Protocol 2, footnote Jc. 

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