5 -End labelling

The terminal dUTP nick end labeling assay (TUNEL reaction) and electrophoretic analysis of DNA/organotin(IV) mixtures allowed the investigation of DNA fragmentation. 

TABLE 7.2 Autoradiogram of the Reductive Cleavage of a 5 -32P End-Labeled 514 bp Restriction Fragment from pBR322 DNA (Ecorl/Rsal) by Cy cl open t />] indoles... 

Maxam, A. M. Gilbert, W. Sequencing end-labeled DNA with base-specific chemical cleavages. Methods Enzymol. 1980, 65, 499-560. 

Fig. 8 Autoradiograms of a denaturing polyacrylamide gel for photooxidation of duplex 35/36 in the presence of BamH I. ODNs 35 (a) and 36 (b) were separately 5 -32P-end labeled and hybridized to a non-labeled complementary strand. Lane 1, Maxam-Gilbert G+A sequencing reactions lane 2, in the absence of BamH I lanes 3-5, BamH I. ODNs in lanes 2-4 were irradiated at 312 nm. All samples except in lane 2 were heated with piperidine. The BamH I site and dCNBPU (X) are shown in bold face. For clarity, the autoradiogram for ODN 36 is shown upside-down...

Figure 31 compares the dynamic structure factors obtained from the crosslinks and the chain ends for two different Q-values. Without any analysis a strong reduction of the cross-link mobility compared to that of the chain end is obvious. A closer inspection also shows that the line-shape of both curves differs. While S(Q,t)/S(Q, 0) from the chain end decays continuously, S(Q,t) from the cross-links appears to decay faster at shorter than at longer times. This difference in line shape is quantified via the line shape parameter p. For the end-labelled chains, p is in close agreement with the p = 1/2 prediction of the... 

Fig. 31. NSE spectra of linear end-labelled PDMS chains (Mw = 5500 g/mol) and of four-functional cross-links at T = 373 K. The solid lines are visual aids. (Reprinted with permission from [84]. Copyright 1988 The American Physical Society, Maryland)...

Hydrolysis of end-labeled 3-hydroxybutyrate oligomers by purified A. faecalis T poly(3HB) depolymerase showed that the enzyme mainly cleaved the second and third ester linkage from the hydroxyl terminus [69]. However, since the enzyme also hydrolyzes cyclic oligomers, the A. faecalis depolymerase has endo-hydrolase activity in addition to exo-hydrolase activity [18, 70]. Results of... 

In order to facilitate analysis of FeBABE produced fragments, the prey protein or biomolecule is labeled at one end with a tag that can be detected after electrophoresis, usually in a transfer blot. The tag can be a fusion tag, such as 6X His, or any other group that can be targeted with an antibody and detected. Alternatively, radiolabels and fluorescent labels have been used with prey molecules, including the use of end-labeled DNA to study where DNA binding proteins dock onto the oligonucleotide sequence. 

The fragments formed by FeBABE fragmentation are analyzed by comparing them to enzymatic or chemical cleavage patterns observed by treatment on the same prey protein. Since the cleaved prey protein is detected by its end-labeled tag, the only fragments detected are those... 

The following protocol assumes that the user has at least two pure proteins (or biomolecules) of known sequences, which are able to interact specifically in solution. One of the two proteins (the prey) is end labeled with a fusion tag or another detectable component and the other protein contains at least one thiol group. All buffers and reagents used in this protocol should be of high purity and contain a very low metal content to prevent nonspecific cleavage reactions. 

The donor-acceptor distance may not be unique, especially when the donor and acceptor are linked by a flexible chain (e.g. end-labeled oligomers of polymethylene or polyethylene oxide, oligopeptides, oligonucleotides, polymer chains). 

In this method the single-stranded DNA fragment to be sequenced is end-labelled by treatment with alkaline phosphatase to remove the 5 phosphate, followed by reaction with 32P-labelled ATP in the presence of polynucleotide kinase, which attaches 32P to the 5 terminal. The labelled DNA fragment is then divided into four aliquots, each of which is treated with a reagent which modifies a specific base as follows. 

TUNEL Assay (Terminal Transferase dUTP Nick End Labelling)... 

TUNEL terminal transferase dUTP nick end labelling... 

Gel electrophoresis of P-end-labelled oligonucleotides irradiated with visible light in the presence of RufTAP)] showed that the principal photochemical product is a less electrophoretically-mobile species [96]. This is consistent with the formation of a photo-adduct and it is clear that the yield of this reaction is... 

Fig. 2. Apoptotic cells fluorescently labeled using the TUNEL assay. Fixed paraffin-embedded rat mammary glands, 4 d postweaning (obtained from Oncor), were labeled using the TUNEL assay to detect apoptotic cells. Reagents were from Oncor s Apoptag Direct In Situ Apoptosis Detection Kit (Fluorescein), which directly incorporates fluorescein-labeled dUTP in the TdT end-labeling reaction. All reagents were used as described by the manufacturer (see Note 1). (Abbreviations are as in text.)...

Zhang, Z. and Galileo, D. (1997) Direct in situ end-labeling for detection of apoptotic cells in tissue sections. Biotechniques 22, 834-836. 

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