Labelling nitrocellulose

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. 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...

Figure 3. E. chrysanthemi cell surface labelling with sulfo-NHS-biotin. After labelling, the proteins were separated by SDS-PAGE, blotted onto nitrocellulose and revealed with PemB-antibodies (A) or with streptavidin-peroxidasc (B). Lane 1 A350 (wild type) lane 2 A837 kdgRy, lane 3 A350/pPME6. An arrowhead indicates the PemB position.

One of the first practical applications for these fluorescent labelled heparins was to examine the heparin binding behavior of different proteins and peptides under study in our laboratories. To this end we used a modification of the dot-blot assay described by Hirose and colleagues (13). F-D labelled heparin (-1 fluorescein/heparin) was radiolabelled with 125Iodine using iodobeads, to a specific activity of approximately 0.5 x 106 cpm/pg. Solutions of proteins with known heparin-binding capacities were dotted on nitrocellulose paper. A series of replicates... 

A Petri dish containing bacterial colonies is blotted with nitrocellulose paper. This transfers a large portion of each colony to the paper, which is saturated with a solution that lyses (breaks open) the cells. The DNA of the lysed colonies is denatured with alkali. The nitrocellulose paper is neutralized, washed, and the paper either baked in an oven or treated with ultraviolet light to immobilize the denatured DNA. The DNA on the paper is hybridized with the labeled probe of interest, and the excess label is washed off. The dried paper is exposed to photographic film and the film developed. The exposed spots on the film can be matched with the colonies on the master plate and colonies picked off for further study. 

The reagent also has been used in a unique tRNA-mediated method of labeling proteins with biotin for nonradioactive detection of cell-free translation products (Kurzchalia et al., 1988), in creating one- and two-step noncompetitive avidin-biotin immunoassays (Vilja, 1991), for immobilizing streptavidin onto solid surfaces using biotinylated carriers with subsequent use in a protein avidin-biotin capture system (Suter and Butler, 1986), and for the detection of DNA on nitrocellulose blots (Leary et al., 1983). 

Leary, J.J., Waldrop, A.A., and Ward, D.C. (1983) Rapid and sensitive colorimetric method for visualizing biotin-labeled DNA probes hybridized to DNA or RNA immobilized on nitrocellulose Bio-blots. Proc. Natl. Acad. Sci. USA 80, 4045 1049. 

Macroarrays [7] Probes are spotted onto nylon, plastic or nitrocellulose solid matrix 8cm by 12cm with approximately 200 to 5000 genes Radioactivity tag labeling phosphorimager detector Passive Clontech Laboratories, Research Genetics... 

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-... 

A related approach is realized in filter binding assays. Here the reaction solution is filtered, e.g., through nitrocellulose where proteins are absorbed, while small molecules can pass. One example of this technique is the quantification of protein bound and free nucleotides (with radioactive labeled ligands). 

Another similar technique to Southern blotting is Northern blotting. Here, instead of DNA fragments, mRNA fragments are probed with a labelled cDNA probe after separation by electrophoresis and transfer to nitrocellulose membranes. Northern blotting is used to detect and quantify mRNA from tissue extracts. 

Figure 13.15 Detection of a specific sequence of DNA by hybridization to a 32P-labelled cDNA probe. DNA is transferred to nitrocellulose and incubated with the probe. After washing, specific binding is visualized by autoradiography. The DNA sequence detected by the probe is present in lanes 2, 3 and 5 but not 1 and 4.

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. 

The specific labeled separated protein fractions blotted on a nitrocellulose membrane or specific immunoflxation-separated protein fractions in polyacrylamide after isoelectric focusing make it possible to detect some additional bands in CSF, i.e., IgM, IgA, free kappa or lambda light chains of specific antibodies (i.e., antiherpes, anti-borrelia, or anti-HIV) (LI, M3). 

DNA in the test sample is denatured to give single-stranded DNA, immobilized on a nitrocellulose or other suitable filter and hybridized with labelled DNA prepared from the host-vector manufacturing system (DNA probes). Although a wide variety of experimental approaches is available, hybridization methods for measurement of host-vector DNA meet the following criteria ... 

FIGU RE 3.7 Visualization by autoradiography of electrophoresed, radiolabeled DNA. After electrophoresis the DNA bands are blotted onto a nitrocellulose membrane, and x-ray film is placed next to the membrane. The positions of labeled DNA bands appear when the film is... 

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