Hrp Hrp

Alkaline phosphatase HRP

Alkaline phosphatase Alkaline phosphatase

4-chloro-1-naphthol (4CN) 3,3'-diaminobenzidine

3,3 ',5,5 ',-tetramethylbenzidine

5-bromo-4-chloro-3-indolyl phosphate/nitro-blue tetrazolium

Luminol/H2O2/p-iodophenol 1-2 dioxetane derivatives Disodium 3-(4-methoxyspiro {1,2-dioxetane-3,2'-(5'-chloro) tricyclo[3.3.1.13,7]decan) 4-yl)-1-phenyl phosphate and derivatives (CSPD, CDP-Star)

Purple precipitate Dark brown precipitate Dark purple stain Dark blue stain

Blue light

Light

Light cannot detect tiny deletions or insertions of nucleotides or single nucleotide differences unless they affect a specific restriction site. For some assays, cross-hybridization may confuse results. These artifacts can be identified by their presence in every lane at a constant size.

Northern (or Western) blots are usually used for analysis of gene expression, although they can also be used to analyze transcript size, transcript processing, and protein modification. For these analyses, especially when estimating expression, it is important to include an internal control to correct for errors in isolation, gel loading, or transfer of samples. The amount of expression is then determined relative to the internal control (Fig. 6-20). In the example shown, the target transcript or protein product is expressed in increasing amounts, left to right. The internal standardized control (lower band) assures that a sample has low expression of target transcript of protein product and that the low signal is not due to technical difficulties.

Array-Based Hybridization Dot/Slot Blots

There are many variations on Southern hybridization methods. In cases where the determination of the size of the target is not required, DNA and RNA can be more quickly analyzed using dot blots or slot blots. These procedures are usually applied to expression, mutation, and amplification/deletion analyses.

For dot or slot blots, the target DNA or RNA is deposited directly on the membrane. Various devices, some

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