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A specific probe is used to locate the presence, and in some cases, quantify the amount of target on the blot.
Probes may be natural or synthetic DNA or RNA. Probes for Southern blots are usually a short synthetic oligonucleotide of at least 25 complementary bases. Probes for Northern blots include complementary DNA (cDNA) synthesized using labeled primers for the target RNA sequence, and in vitro transcribed RNA (riboprobes).
The choice of the label depends on many factors such as sensitivity, quantification requirements, ease of use, and experimental time.
Radioactive labeling, most commonly involving enzymatic incorporation of 32P, 33P, or 35S. Radioactive labeling provides the most sensitive method for detection, allowing detection of 0.01 pg.
Indirect, nonradioactive labeling can involve attaching the probe to an antibody directed against an enzyme. The immobilized probe can then be detected with the enzyme, which breaks down a chemiluminescent or chemifluorescent substrate to produce a light signal. Alternatively the probe can be labeled with biotin, making it detectable by a streptavidin/avidin-enzyme conjugate.
Direct, nonradioactive labeling involves attaching the probe to an enzyme (e.g., alkaline phosphatase or horseradish peroxidase) that can be detected directly using a chemiluminescent or chemifluorescent substrate to produce a light signal.
Probes may also be labeled with fluorophores for fluorescent detection.
|Non-radioactive - Indirect detection||
|Non-radioactive – Direct detection||
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