By Todd H. Otis
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Additional resources for A review of nuclear energy in the United States: hidden power
After passing through the column, the charged solution is sprayed into a mass spectrophotometer. The charged droplets contain fragmented single ions. The peptides are then fragmented and the mass–charge ratios of the fragments are measured. The resulting spectrum is analyzed with a computer program and compared against other known protein sequences within the database in order to determine the sequence of the fragments. Running a sample multiple times using different enzymes that cleave the protein at various regions allows the sequence of the protein to be constructed according to overlaps from the different peptides.
A typical application is for the detection or relative quantification of a specific protein in an isolated tissue sample. ELISA is a technique used for the detection of antibodies or antigens from a sample. Whereas Western blots are run on gels, ELISAs are run on multi-well plates. A basic ELISA consists of affixing an unknown amount of antigen to the surface of a well; serum is a commonly used sample, then an antibody is added that attaches to a specific affixed antigen and finally an enzyme substrate is added for detection.
The enzyme T4 kinase is often used for the addition of labeled radioisotope or similarly labeled phosphate to the 5 termini or end of a DNA fragment. End-labeled synthetic oligonucleotides are useful for numerous applications such Maxam–Gilbert sequencing, DNase footprinting or protection assays, and labeling of DNA fragments as probes. Northern and Southern Blotting Northern and Southern blots are routine methods for the evaluation of specific sequences from RNA or DNA samples. Southern blots get their name from Edwin Southern, the developer of the technique.