Preparative Sodium Dodecyl Sulfate Research Articles

Membrane strip affinity purification of autoantibodies.

A method for affinity purification of autoantibodies from a membrane strip using small volumes of human sera is described. The membrane strip is excised from a western blot containing a target antigen electrophoretically transferred from a sodium dodecyl sulfate (SDS) polyacrylamide gel. This method is a very useful alternative for affinity column chromatography, particularly when the antigen of interest is of low abundance in a HeLa cell extract. The protein mixture is resolved on a preparative SDS polyacrylamide gel and transferred to nitrocellulose membrane. A couple of strips are excised from either side of the blotted membrane and immunoblotted with specific antisera to identify the target band. Then the target band is excised horizontally and used for affinity purification. We have used this procedure to affinity purify antibodies to a 70,000 molecular weight protein derived from HeLa cell extract. A sham band, excised away from the target antigen, was used as a control for sham purification of autoantibodies. The autoantibodies purified in this manner reproduced the multiple nuclear dot anti-nuclear antibody pattern obtained using crude sera from 21 patients without primary biliary cirrhosis or anti-mitochondrial antibody.

Production of monoclonal antibody against alkylhydroperoxide reductase

Development of a stool antigen immunoassay to detect Helicobacter pylori infection requires monoclonal antibody against the specific antigen. Alkylhydroperoxide reductase (AhpC) of Helicobacter pylori has been described as a specific and unique enzyme for H. pylori and therefore, both H. pylori AhpC and Anti-AhpC could be useful in the development of serologic and stool antigen tests, for detecting and monitoring H. pylori infection. The aims of this study were to prepare a monoclonal antibody against AhpC. Accordingly, The isolation and purification of AhpC from H. pylori were attempted by various techniques including ammonium sulfate precipitation, dialysis, preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. Furthermore mice were immunized intraperitoneally with homogenized gel containing the AhpC band of protein extract of H. pylori in sodium dodecyl sulfate- polyacrylamide gel electrophoresis. The monoclonal antibody was produced using the hybridoma technique.One-dimensional preparative gel electrophoresis allows a single and short purification step, the high-resolution capacity of this technique leads to a high level of purity of the enzyme and consequently to a very high specificity of the antibody. The high specificity of antibody was identified by immune blotting in which the antibody reacted with the purified AhpC and whole cell protein extract from H. pylori in addition to the intact cells of H. pylori . This approach is simple, time and cost-saving for preparation of monoclonal antibody against AhpC of H. pylori .

Electroelution of the stained alkyl hydroperoxide reductase of Helicobacter pylori from preparative sodium dodecyl sulfate polyacrylamide gels

Electroelution of the stained alkyl hydroperoxide reductase of Helicobacter pylori from preparative sodium dodecyl sulfate polyacrylamide gels

Production of Polyclonal Antibody Against Alkyl Hydroperoxide Reductase ofHelicobacter pyloriand Its Antigenicity

Stool-antigen detection kits for diagnosis of Helicobacter pylori infection have been widely used because of their full non-invasive nature. Because Helicobacter pylori strains show a distinctive genetic diversity, it is important to find a protein that is a common antigen of various strains and shows a strong immunogenicity for the development of a stool- antigen detection kit. Alkyl hydroperoxide reductase (AhpC) of Helicobacter pylori strongly reacts with the sera of patients with gastritis and peptic ulcer. Therefore, AhpC seems to be an excellent candidate as a target protein for this study. Accordingly, polyclonal antiserum against AhpC was produced in adult New Zealand white rabbits by using AhpC in the gel bands without adding Freund's adjuvant. In addition, isolation and purification of AhpC were perfomed by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and electroelution. In this study, a simple method was used for rapid production of polyclonal antibody against AhpC of H. pylori, which avoids both the long-term AhpC purification and the addition of Freund's adjuvant. One-dimensional preparative gel electrophoresis allows a single and short purification step; the high-resolution capacity of this technique leads to a high level of purity of the protein and consequently to a very high specificity of the antibody. Moreover, this method avoids contamination by other non-specific proteins, which often appear during the purification process by column chromatographic techniques, which may also decrease the purity of the immunogen. The present method is simple, rapid and cost-effective, and also makes it possible to produce antibody for stool-antigen enzyme immunoassay in a short time and at low cost.

An approach for isolating high-molecular-weight glutenin subunit genes using monoclonal antibodies

High-molecular-weight glutenin subunits (HMW-GSs) play an important role in the breadmaking quality of wheat flour. In China, cultivars such as Triticum aestivum 'Xiaoyan No. 6' carrying the 1Bx14 and 1By15 glutenin subunits usually have attributes that result in high-quality bread and noodles. HMW-GS 1Bx14 and 1By15 were isolated by preparative sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and used as an antigen to immunize BALB/c mice. A resulting monoclonal antibody belonging to the IgG1 subclass was shown to bind to all HMW-GSs of Triticum aestivum cultivars, but did not bind to other storage proteins of wheat seeds in a Western blot analysis. After screening a complementary DNA expression library from immature seeds of 'Xiaoyan No. 6' using the monoclonal antibody, the HMW-GS 1By15 gene was isolated and fully sequenced. The deduced amino acid sequence showed an extra stretch of 15 amino acid repeats consisting of a hexapeptide and a nonapeptide in the repetitive domain of this y-type HMW subunit. Bacterial expression of a modified 1By15 gene, in which the coding sequence for the signal peptide was removed and a BamHI site eliminated, gave rise to a protein with mobility identical to that of HMW-GSs extracted from seeds of 'Xiaoyan No. 6' via SDS-PAGE. This approach for isolating genes using specific monoclonal antibody against HMW-GS genes is a good alternative to the extensively used polymerase chain reaction (PCR) technology based on sequence homology of HMW-GSs in wheat and its relatives.

Characterization of Kafirin and Zein Oligomers by Preparative Sodium Dodecyl Sulfate−Polyacrylamide Gel Electrophoresis

Quantitative and qualitative analysis of uncooked zein and kafirin fractions were performed through sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and electrophoretic profiles. Kafirins and zeins present the same oligomer and monomer compositions with the exception of a 66 kDa oligomer that is only present in kafirins. The quantitative analysis showed differences between zein and kafirin. The composition of each oligomer was established via preparative SDS-PAGE. Part of the cooked oligomers resists reduction; the presence of those oligomers could be related to the decrease on protein digestibility with the cooking process.

Identification of the Outer Membrane Porin of Thermus thermophilus HB8: the Channel-Forming Complex Has an Unusually High Molecular Mass and an Extremely Large Single-Channel Conductance

The outer membrane of the thermophilic bacterium Thermus thermophilus was isolated using sucrose step gradient centrifugation. Its detergent extracts contained an ion-permeable channel with an extremely high single-channel conductance of 20 nS in 1 M KCl. The channel protein was purified by preparative sodium dodecyl sulfate (SDS)-polyacylamide gel electrophoresis. It has a high molecular mass of 185 kDa, and its channel-forming ability resists boiling in SDS for 10 min.

Evaluation of the Electroosmotic Medium Pump System for Preparative Disk Gel Electrophoresis

This paper describes an improved electroosmotic elution system for preparative sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) based on the epochal idea of H. V. Tan et al. (Nucleic Acids Res. 1988, 16, 1921–1930). In this elution system, a semipermeable membrane, mounted under the gel terminal end, works as the elution pump as well as the partition of the elution chamber. We refer to this system as the “electroosmotic medium pump system.” Operation of the constructed apparatus (3.6 cm i.d. disk gel column) and resolution of the protein bands were examined by separation of the model protein mixture (bovine serum albumin (BSA), ovalbumin, bovine carbonic anhydrase, soybean trypsin inhibitor) and purification of the membrane protein, dipeptidyl peptidase IV (DPP IV). The Spectra/Por 7 dialysis membrane provided a better flow profile for the elution buffer. The four model proteins of the protein mixture were able to be completely separated from each other and recovered without dilution. The maximum protein concentration of eluate achieved was 93 mg/ml, when applying a single component, BSA fraction V, as a sample. Furthermore, the multifunctional ectoenzyme, DPP IV, was purified in a single step.

Biochemical identification and biophysical characterization of a channel-forming protein from Rhodococcus erythropolis.

Organic solvent extracts of whole cells of the gram-positive bacterium Rhodococcus erythropolis contain a channel-forming protein. It was identified by lipid bilayer experiments and purified to homogeneity by preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). The pure protein had a rather low molecular mass of about 8.4 kDa, as judged by SDS-PAGE. SDS-resistant oligomers with a molecular mass of 67 kDa were also observed, suggesting that the channel is formed by a protein oligomer. The monomer was subjected to partial protein sequencing, and 45 amino acids were resolved. According to the partial sequence, the sequence has no significant homology to known protein sequences. To check whether the channel was indeed localized in the cell wall, the cell wall fraction was separated from the cytoplasmic membrane by sucrose step gradient centrifugation. The highest channel-forming activity was found in the cell wall fraction. The purified protein formed large ion-permeable channels in lipid bilayer membranes with a single-channel conductance of 6.0 nS in 1 M KCl. Zero-current membrane potential measurements with different salts suggested that the channel of R. erythropolis was highly cation selective because of negative charges localized at the channel mouth. The correction of single-channel conductance data for negatively charged point charges and the Renkin correction factor suggested that the diameter of the cell wall channel is about 2.0 nm. The channel-forming properties of the cell wall channel of R. erythropolis were compared with those of other members of the mycolata. These channels have common features because they form large, water-filled channels that contain net point charges.

An alternative for purification of low soluble recombinant hepatitis C virus core protein: preparative two-dimensional electrophoresis.

For isolation of low soluble recombinant full-length (amino acids 1-191) core protein of hepatitis C virus (HCV) overexpressed in Escherichia coli, the advantage of combining two electrophoretic techniques, in comparison with chromatographic separation, is demonstrated. The protein extract was first solubilized in agents compatible with electrophoretic separation. Using preparative liquid phase isoelectric focusing (IEF) the protein of interest was first concentrated within a defined acidic pH range. These fractions were then submitted to preparative sodium dodecyl sulfate - polyacrylamide gel electrophoresis (SDS-PAGE) to isolate the 22 kDa protein. The second-dimensional step allowed the isolation of 2 mg of the purified recombinant HCV core protein (rHCV-C191) from 1.5 g bacterial pellet. This quantity is sufficient to characterize the protein and to perform immunogenicity studies. This procedure of two-dimensional preparative electrophoresis is applicable to a wide range of biological samples and represents an alternative for purification of insoluble proteins.

Purification and Properties of a Highly Thermostable, Sodium Dodecyl Sulfate-Resistant and Stereospecific Proteinase from the Extremely Thermophilic Archaeon Thermococcus stetteri

The cultivation of the extremely thermophilic archaeon Thermococcus stetteri in a dialysis membrane reactor was paralleled by the production of an extremely heat-stable proteinase(s). By applying preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, an SDS-resistant proteinase was purified 67-fold in one step with a yield of 34%. The purified enzyme, which was composed of a single polypeptide chain with a molecular mass of 68 kDa, showed a broad temperature and pH profile (50 to 100(deg)C; pH 5 to 11). The optimal activity with substantial thermal stability was measured with casein at 85(deg)C and pH 8.5 to 9. Inhibition by phenylmethylsulfonyl fluoride and diisopropylfluorophosphate demonstrated that the enzyme was a serine proteinase. The enzyme displayed a relatively narrow substrate specificity, catalyzing the hydrolysis only of N-protected p-nitroanilides or p-nitrophenyl esters of basic (Arg or Lys) or hydrophobic (Phe or Tyr) l-amino acids. l-Phenylglycine amide was also attacked by the proteinase, but with a lower specificity constant. Within the detection limit, no hydrolysis of d-amino acid derivatives was observed. The catalytic efficiency of the enzyme at 80(deg)C (k(infcat)/K(infm) for benzoyl-Arg-p-nitroanilide, 10(sup4)) is the same order of magnitude when compared with that of functionally similar mesophilic enzymes. The proteinase also acts as a transferase, catalyzing the acyl transfer from protected amino acid ester or amide to amino acid amide. The observed thermostability, SDS resistance, relatively narrow substrate specificity, high stereospecificity, and limited catalytic efficiency probably reflect the tighter packing of the thermostable protein molecule and its limited flexibility. This was supported by fluorescence spectra of the enzyme, mainly due to tryptophan residues, in the temperature range of 30 to 90(deg)C. Structural reorganization was observed at temperatures over 100(deg)C. The results obtained could be of relevance for the better understanding of the structure-function relationship of enzymes from extreme thermophiles and suggest possible biotechnological application of the proteinase for resolution of racemic mixtures.

Ultraviolet Shadowing of Proteins in Preparative Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Ultraviolet Shadowing of Proteins in Preparative Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis

Purified Pasteurella haemolytica leukotoxin inducesexpression of inflammatory cytokines from bovine alveolar macrophages

We obtained biologically active purified leukotoxin (Lkt) from Pasteurella haemolytica serotypel, strain 12296 using preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Three species of Lkt of molecular masses 95, 100, and 104 kDa were obtained. Purity of all three species of Lkt was confirmed by analytical SDS-PAGE and Western blot (immunoblot) analysis. Results from the chromogenic Limulus amebocyte lysate assay and silver staining of SDS-PAGE patterns indicated that the preparations were free of contaminating lipopolysaccharide. We then studied the kinetics of TNF α and IL-1 β mRNA expression in bovine alveolar macrophages stimulated with the purified 104 kDa Lkt. Subcytolytic concentrations of Lkt induced TNF α and IL-1 β gene expression and peak induction was observed at a concentration of 1 leukotoxin unit/ml. Both TNF α and IL-1 β mRNA expression were detectable at 1 h after stimulation with 1 leukotoxin unit/ml. The expression peaked at 2 h, steadily declining up to 6 h, and was undetectable by 10 h. Secreted TNF α measured by bioassay peaked at 4–6 h and accumulated at a lesser concentration after 6 h. By contrast, secreted IL-1 peaked at 6 h and decreased significantly by 10 h. The ability of purified Lkt to induce TNF α and IL-1 β gene expression and secretion of bioactive proteins was suppressed by Ca 2+ chelating agents, 5 mM EDTA and 5 mM EGTA, but not polymyxin B. Heat-inactivation of the purified Lkt that had lost its cytocidal property completely abrogated induction of TNF α and IL-1 β gene expression and secretion in bovine AMs. To the best of our knowledge, this is the first published report that describes a procedure to obtain a biologically active form of purified exotoxic leukotoxin from P. haemolytica, and demonstrates a role for Lkt in the induction of inflammatory cytokines in bovine pneumonic pasteurellosis.

Purification of Glycosylphosphatidylinositol-Anchored Proteins by Modified Triton X-114 Partitioning and Preparative Gel Electrophoresis

Triton X-114 phase partitioning has frequently been used to obtain preparations enriched in glycosylphosphatidylinositol (GPI)-anchored proteins and other hydrophobic proteins from crude cellular homogenates. We have developed a new modification of this phase-partitioning technique which allows two distinct GPI-anchored proteins of Tetrahymena mimbres to be separated from other hydrophobic as well as hydrophilic proteins and recovered in ∼90% yield. The unique feature of the new method is a 24-h incubation of the first Triton X-114 extract at −20°C. This improves the partitioning of GPI-anchored proteins into the detergent phase while promoting the aggregation of other hydrophobic proteins. Individual GPI-anchored proteins in the detergent phase are then purified to near homogeneity by one-step preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. By substituting Triton X-114/water mixtures for the conventional SDS buffer used to collect proteins eluting from the preparative gel, the purified GPI-anchored protein can be rapidly concentrated from relatively large volumes of eluate by phase partitioning at 32°C. The method is also effective in separating mammalian GPI-anchored alkaline phosphatase from other proteins. It is likely to be of general utility in characterizing the GPI anchor structures associated with nonabundant and abundant GPI-anchored proteins coexisting within the same cell type.

Isolation of the 36-kD German (Blattella germanica) cockroach allergen using fast protein liquid chromatography.

Cockroach allergens have been established as important sensitizing agents in the induction of asthma. In the present investigation, we identified a 36-kD allergen from German cockroach (Blattella germanica) using fast protein liquid chromatography that reacted in the Bla g II monoclonal assay developed by Pollart et al. Defatted whole body German cockroaches were extracted in phosphate-buffered saline and fractionated using gel filtration (Superdex 75) and anion exchange (MonoQ) chromatography. The 36-kD allergen was isolated from this enriched allergen preparation by excision from modified preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) gels, electroelution, dialysis and lyophilization. SDS-PAGE/Western immunoblot analysis using a serum pool of cockroach-sensitive individuals revealed a single band. The lyophilized 36-kD allergen was then analyzed for amino acid composition and digested for peptide amino acid sequencing.

Development of a Latex Agglutination Test for the Rapid Identification of Vibrio parahaemolyticus

A latex agglutination test for the rapid identification of Vibrio parahaemolyticus has been developed. Two bacterial outer membrane proteins, with molecular weights of 36,000 and 34,000, were obtained by preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis and were subsequently used as antigens for producing antibodies in rabbits. Immunoelectron microscopy demonstrated that the two proteins were present in the outer membrane of the microorganism. Latex particles sensitized with the affinity-purified antibodies were used as a reagent for the rapid identification of the bacterium. Of 173 strains (including 94 isolates of V. parahaemolyticus, 40 isolates of other vibrios and 39 strains of other bacteria) tested, the false-negative and false-positive rates were 1.4 and 3.1%, respectively. Some strains of three vibrios (V. alginolyticus, V. harveyi, and V. mimicus) produced false-positive results. However, no cross-reactions were observed with the latex reagent among the 39 strains (33 species) of other bacteria. It is proposed that suspicious colonies (green or blue) of V. parahaemolyticus on thiosulfate-citrate-bile salts-sucrose agar (TCBS) be subcultured to tryptic soy agar with 3% NaCl for overnight incubation. Cultures grown on tryptic soyagar-3% NaCl may be used for latex agglutination and galactosidase assays. V. parahaemolyticus will be latex positive and galactosidase negative. Under this condition, V. parahaemolyticus can be identified in only one day after suspect colonies were observed on TCBS agar.

Attempts to purify a second cellular receptor for a coxsackievirus B3 variant, CB3-RD from HeLa cells

A coxsackievirus B3 variant, CB3-RD, isolated on rhabdomyosarcoma (RD) cells is known to bind HeLa cells at two different receptor protein sites, HR1 and HR2. Since HR2 occurs in almost 50 fold excess of HR1 in HeLa cells, purification of HR2 was attempted, to obtain its partial N-terminal amino acid sequence and its further characterization. This study describes the purification of HR2 from octylthioglucoside solubilized HeLa cell membranes (HeLa-OTG) by preparative isoelectric focusing (IEF) followed by either preparative sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) or affinity chromatography on immobilized receptor monoclonal antibody, RmcA (RmcA-agarose). IEF of HeLa-OTG showed that both HR2 and HR1 could be well separated by this technique and focused with peak maxima around pH 3.7 and 6.7, respectively. Both RmcA and CB3-RD recognized HR2 as doublet bands (60 kD major polypeptide and a minor 55 kD polypeptide) on electroblots under non-reducing conditions. Preparative SDS-PAGE of the pool of IEF fractions containing HR2 (IEF pool) and simultaneous elution of polypeptides from the bottom of the gel during electrophoresis, is shown to be a useful technique in purifying HR2 with only one contaminating polypeptide (65 kD). However, affinity chromatography of the IEF pool on RmcA-agarose yielded HR2 without any detectable contaminating polypeptide. A quantitative chemiluminescence assay was developed to estimate the amount of HR2 on HeLa cells and in solution, when dot blotted on polyvinylidene difluoride (PVDF) membranes and probed with RmcA. Assays revealed that about 1.2% of the total HR2 present on HeLa cells could be obtained by IEF followed by affinity chromatography. Efforts are continuing to obtain sufficient quantities of purified HR2 for partial N-terminal amino acid sequencing.

Production of Monoclonal Antibodies against the Major Capsid Protein of the Lactococcus Bacteriophage ul36 and Development of an Enzyme-Linked Immunosorbent Assay for Direct Phage Detection in Whey and Milk

The only major structural protein (35 kDa) of the lactococcal small isometric-headed bacteriophage ul36, a member of the P335 species, was isolated from a preparative sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis. Monoclonal antibodies (MAbs) were raised against the denatured 35-kDa protein. Six MAbs were selected and characterized. Western blots (immunoblots) showed that all MAbs recognized the 35 kDa but also a 45 kDa that is in lower concentration in the phage structure. Binding inhibition assays identified five families of MAbs that recognized nonoverlapping epitopes of the 35- and 45-kDa proteins. Immunoelectron microscopy showed that these two proteins are localized within the phage head, therefore indicating that the 35 kDa is a major capsid protein of ul36 and that the 45 kDa is a minor capsid protein. With two MAbs, a sandwich enzyme-linked immunosorbent assay (ELISA) was developed for direct detection of lactococcal phages in whey and milk samples. Whey and milk components, however, interfered with the conduct of the assay. Partial denaturation of milk samples by heat treatment in the presence of SDS and beta-mercaptoethanol removed the masking effect and increased the sensitivity of the assay by 100-fold. With the method used here, 10 PFU/ml were detected by the ELISA within 2 h without any steps to enrich or isolate bacteriophages.

Initiation of Bone Regeneration in Adult Baboons by Osteogenin, a Bone Morphogenetic Protein

Osteogenin, and related bone morphogenetic proteins, induce endochondral bone differentiation through a cascade of events which include formation of cartilage, hypertrophy and calcification of the cartilage, vascular invasion, differentiation of osteoblasts, and formation of bone. These events have been studied in a postnatal model of bone development in rodents. Information concerning the morphogenetic potential of osteogenin in primates is a prerequisite for potential clinical application in man. The efficacy of allogeneic osteogenin in primates was investigated in both extraskeletal and skeletal sites in 19-Chacma baboons (Papio ursinus). Osteogenin was isolated from demineralized baboon bone matrix and purified by chromatography on heparin-Sepharose, hydroxyapatite, and Sephacryl S-200. Protein fractions with a molecular mass range of 26-42 kDa induced cartilage and bone differentiation in the subcutaneous space of rats. Final purification to homogeneity was obtained by electroendosmotic elution from a preparative sodium dodecyl sulphate (SDS) polyacrylamide gel, resulting in a single band on a SDS-polyacrylamide gel with an apparent molecular mass of 30-34 kDa, with biological activity in rats. The osteoinductive potential of osteogenin in primates was tested first in intramuscular sites in baboons and found to be active. The bone regeneration potential was investigated in nonhealing calvarial defects surgically prepared in adult male baboons. Baboon osteogenin induced complete regeneration of the cranial wound. These findings in adult primates establish a primary role for osteogenin in initiation and promotion of osteogenesis, and imply a potential therapeutic application based on cell biology of extracellular matrix-cell interactions.

Characterization of an amylase-binding component of Streptococcus gordonii G9B.

The goal of the present study was to begin characterizing the amylase-binding component(s) on the surface of Streptococcus gordonii G9B. Alkali extracts but not phenol-water extracts of this bacterium inhibited 125I-amylase binding to S. gordonii G9B. To identify the bacterial components involved in amylase binding, the alkali extract was subjected to affinity chromatography on amylase-Sepharose. Immunoblotting with a rabbit antiserum against S. gordonii G9B revealed that a 20-kDa streptococcal component was eluted from the amylase-Sepharose with 1% sodium dodecyl sulfate (SDS), 2 M KSCN, or 0.1 M sodium citrate buffer, pH 4.5. Subsequently, the 20-kDa component was prepared from alkali extracts by electroelution from preparative SDS electrophoresis or by gel filtration chromatography. This component was trypsin sensitive, and an antibody raised against it inhibited the binding of 125I-amylase to S. gordonii G9B. Indirect immunofluorescence microscopy and immunogold electron microscopy demonstrated that both bound amylase and the 20-kDa component were localized to the cell division septum on dividing cells or to polar zones on single cells. In addition, exponentially growing bacteria bound more 125I-amylase than stationary-phase cells did. Collectively, these results suggest that a 20-kDa amylase-binding component is present on the surface of the nascent streptococcal cell wall.