Methods and reagents: Chemiluminescent Detection Methods Methods and reagents is a unique monthly column that highlights current discussions in the newsgroup bionet.molbio.methds-reagnts, available on the Internet. This month's column discusses some tips for using chemiluminescent detection methods. For details on how to partake in the newsgroup, see the accompanying box. Detecting labeled biomolecules, such as DNA, RNA and proteins, is an essential part of everyday lab bench work. Although radioactive isotopes have traditionally been used for this, there is an ever-growing need for researchers to break those traditional bonds. Apart from the obvious concern over health risks involved with constant exposure or radioactive contamination by spills and other mishaps, there are also the cost considerations associated with storage, handling, collection and disposal of radioactive materials. In the past, researchers have been slow to adopt non-radioactive methods because of low sensitivity compared with isotopic detection, and most likely because they often encountered variable results when they were first introduced to the commercial market. For example, initial attempts at using an alkaline phosphatase-based chromogenic detection method proved frustrating. The precipitation of an insoluble blue compound obtained through the interaction of 5-bromo-4-chloro-3-indolylphosphate (BCIP) and nitroblue tetrazolium salt (NBT) onto membranes [1] did not always give the best hands-on experimental results. There's no excuse anymore. Having been thoroughly researched and modified to eliminate the variable precipitation steps, the newer chemiluminescent detection methods are sensitive and dependable enough to challenge conventional radioactive labeling and detection methods. New kits on the block ********************* The most commonly used systems for chemiluminescent detection are: (1) the luminol-based `enhanced' chemiluminescence (ECL) [2-4] catalyzed by horseradish peroxidase (HRP) in the presence of peroxides; and (2) the destruction of dioxetane compounds such as DPP, AMPPD, CSPD or CDP-Star, catalyzed through dephosphorylation by alkaline phosphatase (AP) [5,6]. The breakdown reactions of these two systems each give off light, which is easily detected by imaging onto standard X-ray film. Both HRP- and AP-detection methods can be used in combination with either affinity conjugation systems of streptavidin plus biotin, or digoxigenin plus anti-digoxigenin antibody. One problem remaining with the use of non-radioactive methods is that the materials for the detection are usually only available in kit form. Because the detection substrates comprise approximately 90% of the cost of the entire procedure, and the cost is nearly US$3-5 per Southern or western blot, buying a new kit just for the detection components or substrate is just too expensive. Do-it-yourself kits ******************* Thomas Cameron (cameron@risotto.mit.edu) recently wrote to the methods newsgroup asking about how he might make his own reagents for the ECL reactions, and several people posted their preferred method for preparing homemade chemiluminescent detection solutions. David Shire (david.shire@tls1.elfsanofi.fr) wrote that obtaining good results with the ECL system mainly relies on the purity of the p-iodophenol (PIP) and the luminol. The PIP is needed for enhancing the visible light reaction by acting as a co-factor for peroxide activity toward luminol, rather than participating directly in the luminescence itself. When phenolic enhancers are used in combination with HRP, the level of light is said to be increased about 100-fold (Ref. 7). This is why the reaction has been dubbed `enhanced' chemiluminescence [2] or `ECL' (not to be confused with electrochemiluminescence, which has also been called by the same acronym ECL). David Shire suggests that the cheapest PIP be purchased from Aldrich or Fluka and then sublimed in-house at 85 degrees C under vacuum. He wrote that this is extremely easy to do and results in beautiful white crystals of pure iodophenol as compared to the mucky yellow powder obtained from the chemical supply company. He says that the commercially available apparatus to do this is expensive, but that a cheaper homemade version can be made for little cost. Purifying your own PIP can be dangerous and some recommend simply buying the highest grade of purified PIP instead. Netters warn, however, that not all batches of purified PIP work as well as others, and some have complained about poor results from PIP obtained from various chemical companies. To prepare luminol, David Shire says to suspend 3 g of the cheapest luminol you can find (available form Merck-Schuchard, Fluka or Lancaster Synthesis) in 90 ml of 1-butanol and to add 40 ml of 1M NaOH. After the luminol is dissolved by warming and agitating, 30 ml of methanol is added and the recipient cooled in ice before adding 40 ml of 1M HCl. This should then be left at -20 degrees C for 30 min for precipitation. The crystals are then filtered onto a Whatman No. 1 filter, rinsed with dichloromethane and dried under vacuum. To protect the crystals from light, the bottle or tube should be wrapped with a piece of aluminum foil and placed in a dessicator. The two solutions for the ECL detection protocol are prepared as follows. Solution 1 contains 780 ug ml-1 of luminol and 950 ug ml-1 of PIP of in 0.1M Tris-HCl, pH 9.35. Solution 2 contains 100 ul 30% hydrogen peroxide in 1 l of 0.1M Tris-HCl, pH 9.35. Just before use, equal volumes of each solution are mixed together. One other person wrote that a cocktail composed of 1 ml luminol (4mg ml-1 in DMSO), 1 ml p-iodophenol (1mg ml-1 in DMSO) 0.6 ml of 1M Tris-HCl (pH 7.5), 5 ul of 30% H2O2, and 7.5 ml dH2O works equally as well. Netters say that homemade reagents produce excellent results and cost much less than buying a complete pre-packaged kit. In addition, side-by-side comparisons of homemade solutions with ECL kit components show they work as well or better than the commercial ones. Netters brag that freshly made homemade luminol can even give up to twofold more sensitivity than that bought from companies. It should be mentioned, though, that the use of enhancers for ECL is covered by patent. To save even more money on westerns, some netters say they re-use their primary antibodies up to 15 times without loss of signal. When using biotin-labeled probes, netters report that lower background can be obtained by pre-incubating the membrane in a solution of 1% purified casein as blocking agent, rather than using BLOTTO or non-fat dry milk, which can sometimes contain unwanted biotin. Casein can be partially purified from non-fat dry milk by precipitating it with acetic acid, pH 4.6, and then solubilizing it in 0.1M NaOH (Ref. 8). Furthermore, purification to remove any residual biotin can be done by passing the solution through a streptavidin-agarose column. Spaghetti spooning ****************** Are you having troubles cloning large fragments of DNA after LA-PCR? It might be that the transformation efficiency is lowered as a result of the length of DNA. Recently, one person complained that they have tried a number of different techniques to clone long PCR products over the last year, but have had very little success. In response, one netter suggested using 4 units of DNA gyrase isolated from Micrococcus luteus (topoisomerase II) to wind up the DNA before transformation. In a study on the transformation efficiency of large plasmids approaching 20 kb (Ref. 9), a ninefold increase in transformation efficiency was obtained as compared to DNA not treated with gyrase. This is presumably because of the negative supercoiling induced onto the DNA molecule, making it a more compact structure. This might be just enough help to reduce the background of empty vectors, and thus give a much needed break in locating the desired clone. Although not specifically tested with PCR products, the extra advantage might save people from many attempts at trying to clone large PCR amplicons. Betaine patent update ********************* In response to the recent Methods and reagents column describing the use of betaine for optimizing multiplex and LA-PCR (TiBS 22, 225-226) Thomas Weissensteiner (tweissen@hgmp.mrc.ac.uk) wrote that the patent for adding betaine to polymerase buffers has been issued a number different from that previously reported by him. The correct German Patent Number is DE 4411588 C1. He says that an exclusive license has been sold to Epicentre Technologies, Madison, WI, USA, but that it only pertains to products sold within Germany and has not yet been extended to other countries. It will not be long before others join in though, because a very similar patent has been filed by Genzyme Corporation (United States Patent US 5 545 539). References ********** [1] Leary, J. J., Brigati, D. J. and Ward, D. C. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 4045-4049 [2] Whitehead, T. P. et al. (1983) Nature 305, 158-159 [3] Durrant, I. (1990) Nature 346, 297-298 [4] Durrant, I. et al. (1990) BioTechniques 8, 564-570 [5] Bronstein, I., Voyta, J. C. and Edwards, B. (1989) Anal. Biochem. 180, 95-98 [6] Bronstein, I. et al. (1990) BioTechniques 8, 310-314 [7] Van Dyke, K. and Van Dyke, R. (1990) in Luminescence Immunoassay and Molecular Applications (editors), pp. 61-65, CRC Press [8] Holtke, H. J. et al. (1992) BioTechniques 12, 104-113 [9] Zhixing, Y. and Nahon, J-L. (1995) Nucleic Acids Res. 23, 3353-3354 ******************************************************************************* Any statements made by the author are not meant to advocate the use of a particular commercial product or endorse any company. All opinions are those of the author and do not reflect the opinion of the National Cancer Institute or the National Institutes of Health. Copyright: This manuscript is not copyrighted by Elsevier Publishing Company. However, you may not reproduce any portion for resale or edit the text for redistribution, sale, or otherwise without written permission from the author. You found this at the World Wide Web (WWW) Uniform Resource Locator (URL) ftp://ftp.ncifcrf.gov/pub/methods/TIBS/aug97.txt Any reference to this column must be cited as the following published article: Hengen, P. N. 1997. Methods and reagents: Chemiluminescent Detection Methods. Trends in Biochemical Sciences 22(8):313-314. ******************************************************************************* * Paul N. Hengen, Ph.D. /--------------------------/* * National Cancer Institute |Internet: pnh@ncifcrf.gov |* * Laboratory of Mathematical Biology | Phone: (301) 846-5581 |* * Frederick Cancer Research and Development Center| FAX: (301) 846-5598 |* * Frederick, Maryland 21702-1201 USA /--------------------------/* *******************************************************************************