Blotting methods

Rupp and Locker3 Rat liver Homogenized in a solution containing SDS, and proteinase K Not available, using hybridization including dot-blot method RNA purified from FFPE tissue is suitable for hybridization. 

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-... 

The Western blot method is often used in the analysis of host cell impurities. It can be used to identify a recurring impurity. O Keefe et al. used a Western blot to identify an E. coli protein impurity in the preparation of the recombinant fibroblast growth factor (aFGF).29 By using specific antisera to the E. coli host cell proteins, they were able to isolate the impurity and determine its N-terminus amino acid sequence to confirm its identity. Antibodies could be used to determine the concentration of this impurity in sample preparations. 

Another variant of the hybridization assay is the Northern blot. Here it is RNA, not DNA, that is separated on a slab gel and transferred to a membrane. In the original version of the method, a special chemically treated cellulose membrane was used to hold the RNA, since nitrocellulose does not normally bind RNA. However, conditions have now been found where nitrocellulose will indeed retain RNA molecules. Nylon membranes can also be used. Radiolabeled DNA probes and autoradiography are then employed as above in the Southern blot method. The method is often useful in studying how levels of RNA species in a cell vary with stages of development and differentiation. 

Data from electrophoresis is normally recorded photographically. Densitometry may also be performed on the stained gel or bands may be excised/eluted for further analysis (Mayer et al., 1998). The bands may also be isolated from the gels by blotting methods such as electroblotting (McSweeney et al., 1994 O Malley et al., 2000) or immunoblotting (Addeo et al., 1995 Moio et al., 1992) for further characterization and identification. All of the electrophoretic methods, to a certain extent, provide good quality data. But due to the difficulty in quantitative analysis, very few examples are available on the quantification of protein fractions using... 

In a sequence of events reminiscent of the Southern blot evolution into the Northern blot method, arrays of immobilized complementary DNA (cDNA) and expressed sequence tags have emerged on the heels of... 

Okamura, H., Sigal, C. T., Alland, L., and Resh, M. D. (1995) Rapid high-resolution western blotting. Methods Enzymol. 254, 535-550. 

In all these methods proteins are transferred onto one membrane and both total protein staining and immunostaining are performed on the same membrane. Double-replica blotting methods have also been developed to obtain a membrane with all the proteins stained and that is an almost identical copy of the immunostained one. The first of its kind was described by Johansson (66) who found that by chang-... 

Neumann, H. and Mullner, S. (1998) Two replica blotting methods for fast immunological analysis of common proteins in two-dimensional electrophoresis. Electrophoresis 19, 752-757. 

Cheng H, Rudick MJ (1991) A membrane blotting method for following the time course of protein radioiodination background references using IODO-BEADS, Anal Biochem 198 191-193... 

Nishimura and co-workers have developed the catch-and-release strategy between solid-phase and water-soluble polymer supports, the so called pol)uner blotting method , which allows for the rapid and efficient synthesis of glycopeptides [76]. The method involves (i) conven-... 

Cohen MD, Miller CA, Xu LS, et al. 1990. A blotting method for monitoring the formation of chemically induced DNA-protein complexes. Anal Biochem 186 1-7. 

In addition to the detection of antigens and antibodies, EIA will, undoubtedly, play an increasingly important role in molecular biology. For example, the bio-blot method (Leary et al., 1983) for the detection of DNA-DNA or DNA-RNA duplexes on nitrocellulose membranes offers important advantages over conventional procedures in which radioactive probes are used and autoradiographic detection. In this method, biotinylated DNA probes are prepared by nick translation (Rigby et al., 1977) in the presence of biotinylated analogs and hybridized with the DNA or RNA on filters. Biotin is then detected by avidin-labeled enzyme (Section 3.1). 

Fast blot methods to minimize nucleic acid extraction and immobilization steps have been developed. Those with nylon as a solid phase can take advantage of the ability of NaOH to dissociate cells, denature DNA and immobilize DNA. Nitrocellulose membranes have a lower binding capacity and co-immobilization of nucleic acid and protein from neutral solutions can be a problem. Bresser et al. (1983) used hot concentrated Nal to inhibit protein immobilization, to denature DNA and to irreversibly bind the nucleic acid to nitrocellulose (no baking required). This method can also be used for RNA. About 10 cells are minimally required for a unique DNA sequence, whereas > 0.01% of total mRNA can be detected by the Nal methods. 

Both genes reach a peak in expression at around 13 DPF and then decline to very low levels by 25 DPF with HalO mRNA being somewhat more prevalent at 25 DPF than that of Ha5. Both are undetectable in dry seed by RNA gel blot analysis (data not shown). Since the gel blot method gave more accurate quantitative data we continued to use it for the analysis of the expression pattern of other genes of interest. 

Layfield LJ, Willmore-Payne G, Shimada H, Holden JA. Assessment of NMYC amplification a comparison of FISH, quantitative PCR monoplexing and traditional blotting methods used with formalin-fixed, paraffin-embedded neuroblastomas. Anal Quant Cytol Histol. 2005 27 5-14. 

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