Limitations of comparative detection of proteins via epitope tagging.

glass plate) was covered by Saran wrap, labeled by hot ink, and exposed 10 min to autoradiography film. The region corresponding to the fragment of interest was cut with a scalpel, the Saran wrap was removed, and the sandwich with the piece of the DEAE paper and Whatman 3MM paper was prepared as for unlabeled DNA fragment. The rest of the procedure was identical to that for the unlabeled DNA fragment. The method described for isolation of DNA fragments from both agarose and polyacrylamide gels is efficient, simple, and cheap. The yield of the method was about 80–90% as measured after isolation of Plabeled DNA fragments from 100 bp to 4 kb. We have found a similar yield using low-melting-point agarose. The fragments could be used for ligation, digestion FIG. 1. Comparison of yields of DNA fragments isolated by DEAE paper and several other methods. One microgram of the FX174 DNA cut with HaeIII was loaded into three lanes of 1.4% agarose gel in TAE with 0.5 mg/ml ethidium bromide (1). After a short electrophoresis (DNA samples just entered the gel, bromphenol blue was about 5 mm from the slots), the pieces of the gel containing all DNA fragments from two lanes were removed, and DNA was isolated by commercial kits for isolating DNA fragments from agarose gels. The third lane was used for isolating DNA fragments by DEAE paper. A slot was cut in front of the DNA fragments in gel, a piece of DEAE paper was inserted into the slot, and electrophoresis was continued for 30 min, until all the fragments were bound on the DEAE paper. The fragments were isolated from the DEAE paper as described in the text. The fragments isolated by the particular methods were electrophoresed on the 2% agarose gel. Lanes: 1, 1 mg of the FX174 NA cut with HaeIII; 2, the DNA fragments isolated by QIAquick olumn (Qiagen); 3, the modification of the GeneClean technique (2); , the DNA fragments isolated by DEAE paper. polynucleotide kinase or Klenow fragments of DNA polymerase I, respectively, or random-primed labeling. The glycogen present in the fragment did not interfere with any reaction used. A comparison of the method with the two commercial techniques (GeneClean and QIAquick) is exemplified in Fig. 1.