Nitrocellulose filter

Fig. 1. Southern blot analysis of DNA showing (a) step 1, an agarose gel containing separated restriction fragments of DNA, denoted by (—), which is immersed in NaOH to denature the double-stranded stmcture of DNA, and then transferred by capillary flow to a nitrocellulose filter. In step 2, the bound DNA is allowed to hybridize to a labeled nucleic acid probe, and the unbound probe is washed off In step 3, the filter is placed into contact with x-ray film resulting in (b) bands of exposure on the film which are detected after development and correspond to regions where the restriction fragment is...

Transfer (blot) gel to nitrocellulose filter using Soudiern blot technique... 

FIGURE 5. Assay for Met(0)-peptide reductase. The assay for Met(OJ-peptide reductase is carried out in a two-step incubation. The first incubation mixture contains Met(OJ-L12, DTT and the Met(OJ-peptide reductase. At the end of this incubation, purified L12 transacetylase and [3H]acetylCoA are added and the amount of radioactivity incorporated into L7 is determined by its ability to be retained on a nitrocellulose filter. The latter represents the amount of Met(0)-L12 reduced since the oxidized protein is not a substrate for the transacetylase. 

Northern blot A method for transferring RNA from an agarose gel to a nitrocellulose filter, on which the RNA can be detected by a suitable probe. 

In 1987, CL started to be applied in DNA hybridization assays as an alternative to the use of radioactive tags. These assays are based on the specificity of a binding process that of DNA strands for each other. An unknown DNA can be identified with the Southern blot method in which the strands of the analyte are separated and allowed to interact with labeled probe DNA strands on nitrocellulose filter paper. If the label on the probe is detected, the DNA can be identified and, in some cases, quantitated. Conventionally, radioactive tags were used be-... 

If a single-stranded DNA molecule is placed with a complementary single DNA sequence the two molecules will hybridize. This hybridization forms the basis of a number of very powerful techniques for detecting and quantifying specific nucleic acid sequences. The hybridization may be carried out either in solution or more commonly with the DNA immobilized on nitrocellulose filters. The complementary DNA sequence is known as a cDNA probe. Probes for a large number of important nucleic acid sequences are now available. 

The origin of the microarray or biochip can be traced to a seminal publication by Edwin Southern over 30 years ago. Southern described a method by which DNA could be attached to a solid support following electrophoresis and interrogated for sequences of interest by hybridization with a complementary DNA sequence (16). The complementary DNA sequence, termed a probe, was labeled with either a radioactive or a fluorescent marker and hybridized to the DNA target sample, which was immobilized on a sohd support, such as a nitrocellulose filter membrane. 

Ge (2000) carefully designed a universal protein array (UFA) system based on the use of various transcription factors, their activators, and cofactors as probes. A total of 48 different, highly purified factors were used to create the UFA on nitrocellulose filters. Frotein-protein interactions of various binding affinities could be assessed using different ionic strength buffers (e.g., 100 mM KCl vs. 1000 mM KCl). The relative binding of radiolabeled ( T) GST-K-p52 proteins to various transcription factors was studied. 

The first two rounds are performed under low stringency conditions to enhance RNA-protein binding and to avoid early depletion of sequences present in the SELEX RNA pool. For SELEX cycles 1-3 a nitrocellulose-filter binding assay is used to separate receptor-bound from free aptamers. Beginning from SELEX cycle 4, the nitrocellulose-filter binding and a gel-shifr selection step are employed as two consecutive selection processes (see Note 2). 

To perform SELEX experiment using nitrocellulose-filter binding, prepare filtration unit by preincubating a nitrocellulose sheet in incubation buffer. 

Cut out the pieces of the nitrocellulose filters that cover the used wells of the filtration unit. 

Phenol- and chloroform-extract and ethanol precipitate the recovered RNA (see Subheading 3.7). The RNA is reverse transcribed, purified, and precipitated as detailed in Subheadings 3.6 (25) and 3.7, and then RNA is used for the nitrocellulose filter selection step and cocaine displacement of nAChR-bound RNA molecules (Fig. 2). 

Fig. 2. Alternation ot gel-shitt and filter-binding selection steps Target-bound and unbound radiolabeled RNA aptamers are separated by polyacrylamide gel electrophoresis, visualized by autoradiography, purified from the gel, and used for the subsequent nitrocellulose-filter binding selection step. The experiments are earned out in the presence (-i-) and absence (-) of target protein using the SELEX cycles 0 (control), 3, and 7. The figure illustrates the increase of binding affinity of selected RNA pools, seen as augmented quantity of RNA retained together with the receptor protein at the top of the gel (modified from ref. (8)).

Screen selected colonies for desired DNA fragment. In one procedure a small sample from each of the selected colonies is placed onto spots on a nitrocellulose filter. Several colonies can be placed on one filter and the bacteria lysed, hybridized with a radio-... 

Nitrocellulose filters 205 Non-Arrhenius kinetics 555, 598 Nonbonded interactions 325-332 Nondisruptive deletion 426, 560 Nonequilibrium dialysis 202 Nonproductive binding 114-118, 371-372... 

Filters To filter the working solutions from above, use 0 45-pm nitrocellulose filters (e g, Millex-HA, Millipore)... 

For consistent results, the gold stain should be prepared fresh for each batch of nitrocellulose filters. The gold stain is prepared essentially as described by Righetti et al. (3), with some modifications (4) (see also, Chapter 29). 

Nitrocellulose filters (82-mm diameter, BA 85) are obtained from Schleicher Schuell (Keene, NH) (Products from other suppliers may be acceptable.)... 

Use a soft lead pencil to label nitrocellulose filters with a hash maik and letter or number on one edge to allow subsequent identification and orientation (see Note 1)... 

Fig. 1. Specific identification off. coli containing plasmid pBR322. Approximately 225 colonics, consisting of a 10 1 mixture of plasmid-frec and plasmid-containing cells, was grown on a nitrocellulose filter. The filterwas subjected to the lysis protocol described here, followed by a hybridization with biotinylated pBR322. Sites of positive hybridization were detected by means of streptavidin and alkaline phosphatase. The dark sites correspond to colonies harboring pBR322. Plasmid-free cells give the faint signals present at numerous sites on the filter.

The ethanol concentration in step 4 of the lysis protocol is a w/w concentration Ethanol solutions made up v/v, or otherwise in excess of 90% w/w, exceed the ethanol tolerance limits of some batches of nitrocellulose. Filters washed in such solutions may become brittle and be reduced nearly to powder by the end of the hybridization-color assay procedure. The appropriate solution can be made from 100% ethanol. 

Transfer the DNA probes to nitrocellulose filter by Southern transfer (23) Small fragments normally transfer within 4-6 h. 

While the plates are incubating at 42°C, soak the nitrocellulose filters in 10 mM IPTG for 15 min. This may be carried out in one container, but ensure that all of the filters are properly wetted. Allow the filters to dry at room temperature for 1 h on Whatman 3 MM paper When the filters are dry, number the edges of the filters with a ballpoint pen. 

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