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138 BIOCHEMICAL SOCIETY TRANSACTIONS Franke, W.W.( 1 987)Cell48.3-4 Gerace, L. ( 1 986)Trends Biochern. Sci. 11,443-446 Gruenbaum, Y.,Landesman, Y., Drees, B., Bare, J. W., Saumweber, H., Paddy, M., Sedalt, J. W., Smith, D. E., Benton, B. & Fisher, P. A. ( 1988)J. Cell Biol. in the press Jentoft, N. & Dearborn, D. G. (1979)J. B i d . Chern. 254, 4359-4365 Krohne, G. & Benavente, R. ( 1986)Exp. Cell Res. 162,1 - I0 Laemmli, U. K. ( 1 970)Nature (London)227,680-685 March, S.C., Parikh, I. & Cuatrecasas, P. ( 1974)Anal. Biochern. 60, 149-I 5 2 Moss, B. & Rosenblum. E. N. (1972)J. Biol. C'hern. 247, 5194-5198 Olmsted,J.B.(198l)J.Biol.Chern.256,11955-11957 Risau, W.,Saumweber, H. & Symmons, P. ( 1 98 I ) Exp. Cell Res. 133,47-54 Smith, D. E. & Fisher, P. A. ( 1984)J . Cell B i d . 99.20-28 Smith, D. E., Gruenbaum, Y., Berrios, M. & Fisher, P. A. (1987)J. Cell Biol. 105.771-790 Received 10 October I987 Protein-blot analysis of receptor-ligand interactions JONATHAN M. GERSHONI Department of Biophysics, The Weizmann Institute of Science, Rehovot 76100, Rehovot, Israel. Protein blotting was originally introduced as a solid-phase immunoassay (Gershoni & Palade, 1983; Towbin & Gordon, 1984). However, this technique can also be employed for the analysis of ligand-receptor interactions. In principle, a protein sample containing a receptor is resolved by gel electrophoresis and then blotted to an immobilizing matrix, such as a nitrocellulose membrane filter, as would be performed for immunoblotting. The blot is subsequently reacted with the corresponding ligand rather than a solution containing a particular immunoglobulin. The ligand-receptor complex is then detected, usually via autoradiography when the probe can be radioactively labelled. Whereas this corollary to blotting appears to be straightforward, there are a number of basic points that can jeopardize the chances of obtaining good results. Ligand overlay requires that binding capacity be maintained in the blotted counterpart. In immunodetection, the probe is a large, intact, native protein complex designed to recognize a distinct and small immobilized determinant. In the case of receptor detection, the probe might be extremely small and affinity and selectivity are expected of the protein which has undergone gel electrophoresis and blotting. These procedures are usually denaturative and so the dissociation and unfolding of the receptor components might readily destroy any quaternary and tertiary configurations required for the integrity of the ligand binding site. Another issue which should be considered is the detectability of the probe. Where iodinated ligands can be obtained, demonstration of a complex on the blot is easy. Many ligands, however, which are quite adequate for binding assays, are or lJC-labelled and are thus less convenient for our purpose. Nonetheless, in spite of all the above apprehensions, there are quite a few systems for which protein blotting and ligand overlay have been very useful (for recent reviews see Gershoni, 19876, 1988). A case in point is the nicotinic acetylcholine receptor ( AChR). " H - The nicotinic acetylcholine receptor This receptor is a multimeric glycoprotein complex ( a , P y S ) situated on the post-synaptic side of the neuro- muscular junction. The receptor functions as a ligandregulated ion-channel and by binding the agonist, acetylcholine, it elicits membrane depolarization which leads to muscle contraction. The antagonist, a-bungarotoxin (BTX), Abbreviations used: AChR. acetylcholine receptor; BTX. a-bungarotoxin. competes for the acetylcholine-binding site with an affinity of K,= l o - " M. a-Bungarotoxin is a short peptide (74 amino acid residues), derived from snake venom and is easily iodinated (Popot & Changeux, 1984). Protein blots have been prepared from crude membrane preparations containing the AChR. These were then probed with '251-labelled-BTX and autoradiographed. Only the asubunit of the AChR was found to bind BTX although the affinity of the complex formed (Kd= lo-' M ) was markedly reduced as compared with that of intact receptor. Systematic analysis of the BTX binding to blotted a-subunits has allowed the precise identification of the toxin-binding site. Protein blots of proteolysed a-subunits were probed with a variety of different reagents. The toxin was used to reveal the fragment containing the ligand-binding site. Concanavalin A was employed to detect the presence of residue Asn-14 1, the only asparagine which can be N-glycosylated. Sequencespecific antibodies prepared against synthetic peptides were used for the final identification of the various proteolytic fragments generated (Gershoni et al., 1983; Wilson et ul., 1984; Neumann etal., 1985, 1986). In addition, protein blotting has been used to determine the disulphide arrangement of the a-subunit. The AChR was alkylated with a biotinylated maleimide (Bayer et al., 1985) and the SH-containing fragments derived from such receptors were identified using I -51-labelled-avidin overlay of blots (Mosckovitz & Gershoni, 1988). Protein blotting has been extremely important in the study of the structure and function of the native AChR. Currently this approach is being used for the study of bacterially expressed BTX-binding sites as well (Gershoni et al., 1987a). Once the binding site within the a-subunit had been identified, subclones of the complementary DNA of the a-subunit were prepared using the bacterial expression vector pATH2. Escherichia coli transformants produced a fusion protein which binds BTX. Subjecting these clones to recombinant DNA manipulation has allowed the binding-site to be characterized and the specific contribution of individual residues is being ascertained. Most exciting is the fact that the bacterially expressed binding sites have proven to have possible clinical value. These 'mimic binding sites' act as molecular decoys ('decoyants') and can intercept the toxin in the blood stream. Mice have been injected with the decoyant or a placebo and then challenged with neurotoxins. Those which received the decoyant have a 3-4-fold better chance of survival as compared with the controls. Thus by systematic blot analysis of the AChR, not only has the binding site been mapped, this study has led to a novel concept for possible drug design, namely: decoyance. The success of this research has largely been due to the amenability of the BTX/AChR system to blot procedures. Some practical considerations of ligand overlay are discussed with the hope that this may be helpful. 1988 624th MEETING, DUBLIN 134, I’ructicul cotisiderutiori.~ In testing whether o r not a particular system can be subjected to blot analysis a numbcr of preliminary experiments and parameters should be taken into account. (i) Dot blot. Before attempting a gel-blot, dot-blotting should be performed. In doing so, the conditions are determined for an optimal signal-to-noise ratio. In essence, the protein applied as a dot should contain an intact receptor. Probing such dots allows the verification of the suitability of the probe and detection system. Other factors such as the quench solution and washing conditions can be established. (ii) Sunnple prepurutiori. In the dot-blot experiment the receptor is left unperturbed. Most electrophoresis systems require SDS denaturation of the sample. However. a good amount of ‘over-kill‘ is often exerted. The sample should not be boiled, if possible detergent concentration should be kept to a minimum (0.1%) and the effect o f reducing agents should be cvaluated. (iii) Gel electrophoresis. A point which may have an effect is the time course of running the gel. We often want to obtain results as fast as possible and thus run gels at high currents causing unnecessary joule-heating. Running the gel at lower currents is preferable. In addition, usc o f lithium dodecyl sulphate and running the gel at 4°C may be helpful. (iv) Blottirg. Thc buffer composition in blotting may have effects on the conformation o f the protein being blotted. Methanol should be avoided as this tends to ‘fix’ the protein. Whereas electroblotting is used routinely in our studies, blotting by diffusion might provide time for the sample to renature if such renaturation is possible. As to renaturation, pretreatment of the gel before blotting and post-treatment of the blot prior to probing can be beneficial. (v) Processing. The affinity of a ligand for a blotted receptor constituent may be much less than that observed for the native system. Therefore, washing in ice-cold buffer and reducing processing times might be necessary. Once the ligand is bound and excess probe is removed some sort of fixation or cross-linking can be attempted. J.G. thanks Rachel Samuel for preparing this manuscript. This research has been supported by the Yeda Foundation. the Israel Society for Psychobiology, the Minerva Foundation and the Muscular Dystrophy Foundation. Bayer, E. A,, Zalis, M. G. & Wilchek, M. (1985, Ancrl. Hioc.hetn. 149,520-536 Gershoni, J. M. & Palade. G. E. ( 1983) Arid. Hioc.hrm. 131. I - I5 Gershoni, J. M., Hawrot, E. & Lentz, T. L. ( 19x3) /’roc. Nut/. Acud. Sci. U.S.A.80,4973-4977 Gershoni, J. M. ( 1 9 8 7 ~ )/’roc. Nut/. Accrd. .Sci. (1.S.A. 84. 43 18-432 1 Gershoni. J . M. ( 19X7h I in Advunc,c,.\ in I./(,c,lrophorc,.\/.\ (C’hrambach, A,, Dunn, M. & Radola. B. J., eds.), vol. I. VCH Verlagsgesellschaft, Weinheim, FRG, in the press Gershoni, J. M. (1988) in Methods oJ’Hioc~hemiculAnulysis (Glick, D., ed.), vol. 33, John Wiley, New York, in the pre\\ Mosckovitz, R. & Gershoni, J. M. ( 1988)J . H i o l . <’hem.in the press Neumann, D., Gershoni, J. M., Fridkin, M. & Fuchs. S. ( 1985) I’roc. Narl. Acad. Sci. U.S.A.82. 3490-3493 Neumann, D., Barchan, D., Safran, A,, Gershoni, J. M. & Fuchs, S. (1986) Proc. NU^. Acud. Sci. U.S.A.83. 3008-30 1 1 Popot, J.-L. & Changeux, J.-P. ( 1 984) I’hysiol. Rev. 6, I 162- I239 Towbin, H . & Gordon. J. ( 1984) J . Immunol. Meihod.v 72. 3 13-340 Wilson, P. T., Gershoni, J. M., Hawrot, E. & Lentz, T. L. (1984) I’roc. Natl. Acud. Sci. U.S.A. 81, 2 5 5 3 - 2 5 5 7 Received 3 November 1987 Non-radioactive probes in Southern blots ALAN D. B. MALCOLM and HERMIA FIGUEIREDO l)epurttnerit of’Riochernistty, C’haritig C’ross arid Westmitister Medicd School, Fiilhuni I’uluce Roud. Lotidori Wh 8RFs U.K . The use o f DNA probes as diagnostic reagents is becoming increasingly common [ 1 I. Thus they are able to diagnose with great precision and sensitivity infectious (e.g. viruses) (2, 31, inherited (e.g. sickle cell disease) 14, 51 and acquired (e.g. somatic cell changes leading to tumours) diseases [6]. DNA probes have several advantages over the more conventional biochemical techniques for the investigation of disease. Thus the rules by which a DNA probe recognizes its complementary partner have been well understood since the days of Watson and Crick. This is in complete contrast to the rules governing the interaction between an enzyme and a substrate or between an antibody and an antigen. In one or two individual cases we have a good understanding, but no general set of rules is yet available. A consequence of this is that it is possible both theoretically and in practice to design a probe specific for any particular purpose [ 71. A further advantage which nucleic acids have is that all nucleic acid molecules are chemically and physically very similar to each other in being long repeating negatively charged polymers. This means that they all display similar behaviour in response t o physical and chemical changes, and makes dealing with them much more predictable (and hence reproducible) than dealing with, say, enzymes or antibodies. The third advantage which nucleic acids have is that it is possible to detect a particular genome (abnormal o r normal) even in the absence of the gene product for which it codes. Again this is in contrast to the use of enzymic methods or Vol. 16 antibody methods where transcription and translation must both have taken place. Thus sickle cell disease can be diagnosed it1 liter0 [ 8 ] and latent viruses can be diagnosed in tissues before the disease has taken hold 191. Even more remarkably, DNA probes can be used which are not even causally linked to the disease at all. Thus restriction fragment length polymorphisms can be detected where the base change(s) which give rise to the restriction fragment length polymorphism is genetically linked t o the locus causing the disease but does not itself give rise to it [ 101. The tremendous power of DNA probe technology has not surprisingly, therefore, given rise to a wide range of technologies to enable these advantages to be exploited. Southern blotting [ 1 11 allows the transfer of DNA fragments from a gel after electrophoresis on to a suitable membrane such as cellulose nitrate or modified nylon. Spot blots and slot blots simply employ a manifold to allow a sample to be spotted on to the membrane which can then be immobilized by baking [12]. In all these cases, the sample is immobilized to a membrane and after denaturation is then hybridized t o a suitable probe. A further advantage which nucleic acids have over other approaches is the wide range of methods available to produce a labelled probe. Enzymes exist which allow us to perform nick translation [ 131, random oligonucleotide priming [ 141, polynucleotide kinase labelling [ 1.5I and the addition of tails using terminal deoxynucleotidyltransferase [ 161; reverse transcriptase allows us to make a labelled DNA copy from an RNA template [ 171. The chemistry of oligonucleotide synthesis plus chemical methods for labelling DNA can all be added to this list [18]. A wide variety of