Reaction of monoclonal antibodies with plasma membrane proteins after binding on nitrocellulose: renaturation of antigenic sites and reduction of nonspecific antibody binding

The immunochemical reaction of monoclonal antibodies directed against native membrane proteins was investigated after their separation in sodium dodecyl sulfate polyacrylamide gels and electrotransfer to nitrocellulose. Nonspecific binding of antibodies to membrane proteins, which was increased by beta-mercaptoethanol treatment or heat denaturation of the antibodies, could be significantly reduced if 1 M D-glucose plus 10% (v/v) glycerol was added during the incubation with the antibodies. It was found that specific antibody binding was drastically reduced by SDS treatment of the membrane proteins. During the electrotransfer to nitrocellulose and the simultaneous removal of SDS, some increase in antibody binding was observed. Considerable renaturation of antigenic sites in the blotted proteins could be induced if the nitrocellulose blots were incubated for 16 h at 37 degrees C in phosphate-buffered saline. With the introduction of both modifications, the renaturation step, and the addition of D-glucose and glycerol to reduce nonspecific antibody binding, the immunoblot technique may be successfully applied to detect conformational antibodies against membrane proteins.     

Detection of low-molecular-weight polypeptides on nitrocellulose with monoclonal antibodies

An immunoblotting method to detect low-molecular-weight peptides with monoclonal antibodies that normally fail to demonstrate immunoreactivity using conventional blotting techniques is described. Detection of neurophysin, insulin, calcitonin, vasopressin, and beta-endorphin electroblotted on nitrocellulose membranes was optimized after introducing four modifications into the conventional procedure. These include renaturing the gels after sodium dodecyl sulfate electrophoresis, electroblotting the renatured gels in basic transfer buffer, fixing and/or heating the blots, and using avidin/alkaline phosphatase conjugates for antigen/antibody detection. This technique likely enables the denatured peptides to regain their native conformation and, therefore, restores antigenicity and recognition by highly structural specific monoclonal antibodies. Although the most dramatic improvement with this technique is with monoclonal antibodies, a modest improvement in sensitivity can be obtained when immunoblots are probed with polyclonal antibodies. The high resolution of this system will be useful in probing blots of partial proteolytic digests of proteins with both monoclonal and polyclonal antibodies.     

Immunochemical Studies of Bovine and Human Choline-O-Acetyltransferase Using Monoclonal Antibodie

Abstract: Immunochemical properties of bovine and human choline acetyltransferase (ChAT, EC 2.3.1.6, acetyl-CoA:choline- O -acetyltransferase) were studied using six monoclonal antibodies (AB1, AB5, AB6, AB7, AB8, and AB9) reactive with the enzyme. All antibodies except AB1 bound specifically to two proteins of 68,000 and 70,000 MW on "Western" blots of sodium dodecyl sulfate-polyacrylamide gels containing human or bovine ChAT. The enzyme was specifically absorbed to immobilized antibody and could not be eluted by low pH and/or high salt concentrations, although the enzyme retained activity on the immunoabsorbent. Pure bovine enzyme consisting of the same two proteins as seen in the Western blotting studies was eluted from immobilized AB1 in the presence of sodium dodecyl sulfate. Although active enzyme could not be eluted from immobilized antibodies by standard conditions, various combinations of free and immobilized antibodies were effective in competing off bound enzyme. Free antibody AB1 quantitatively eluted the active enzyme from immobilized AB1. The different capacities of the antibodies to elute enzyme from various immunoabsorbents reflect interesting properties of both the enzyme and the antibodies.     

Antigen retrieval in cryostat tissue sections and cultured cells by treatment with sodium dodecyl sulfate (SDS)

A simple method for antigen retrieval in tissue sections and cell cultures is described. Because many antibodies recognize denatured proteins on western blots, but are poorly reactive by immunocytochemistry, the effect of applying sodium dodecyl sulfate (SDS) to cryostat sections of tissues and to cell cultures prior to immunostaining was examined. In many cases, a 5-min pretreatment with 1% SDS produced a dramatic increase in staining intensity by indirect immunofluorescence. Among the antibodies tested that showed a positive effect of SDS were an anti-Na/K-ATPase monoclonal antibody, an anti-AE1/2 anion exchanger polyclonal antipeptide antibody, a monoclonal anti-caveolin antibody, and an anti-rab4 monoclonal antibody. In other cases, including antibodies against gp330, aquaporin 1, and aquaporin 2, no effect of SDS was detected. The results show that SDS treatment can be used as a simple method of antigen retrieval in cryostat sections and on cultured cells. In some cases, antigens were not detectable without pretreatment with SDS.     

Analysis of sperm antigens by sodium dodecyl sulfate gel/protein blot radioimmunobinding method

A radioimmunobinding method based on the blotting of renatured proteins from sodium dodecyl sulfate gels on to nitrocellulose filter papers was developed to analyze the sperm antigens that elicit serum anti-sperm antibodies. In rabbits, serum anti-sperm antibodies were raised by immunization with homologous epididymal spermatozoa mixed with complete Freund's adjuvant. The raised antisera from either male or female rabbits were shown to react with three major sperm protein bands on sodium dodecyl sulfate gels with the corresponding molecular weights of about 70,000 ± 5000, 14,000, and 13,000, respectively. In humans, the monoclonal antibodies against human sperm were raised by a hybridoma technique. Out of six independent hybrid cell lines that were generated, three of them were shown to secrete immunoglobulins that react with the same two protein bands on sodium dodecyl sulfate gels, which have the approximate molecular weight of 10,000. The same procedure was also used to analyze human serum samples that were shown to contain anti-sperm antibodies by the known techniques. Unique sperm antigens that elicit anti-sperm antibodies in humans were identified and correlated. The results of this study suggest that sodium dodecyl sulfate gel/protein blot radioimmunobinding method may be a sensitive and useful tool for the study of sperm antigens that elicit autoimmune responses and their association with human infertility.     

Blotting intact immunoglobulins and other high-molecular-weight proteins after composite agarose-polyacrylamide gel electrophoresis

A composite agarose-polyacrylamide gel containing urea and sodium dodecyl sulfate reliably resolved unreduced human immunoglobulins according to their molecular weight. Intact immunoglobulins and a number of other macromolecules were readily transferred to nitrocellulose paper by either capillary or electrophoretic blotting, although the latter technique was more effective. Conventional antigen probing as well as immobilized antibody studies can be performed on the nitrocellulose transfers.     

125I-wheat germ agglutinin blotting: increased sensitivity with polyvinylpyrrolidone quenching and periodate oxidation/reductive phenylamination

Two substantial improvements in sensitivity in the identification of 125I-wheat germ agglutinin-binding glycoproteins on nitrocellulose blots of sodium dodecyl sulfate-polyacrylamide gels are reported. The major improvement in sensitivity (about 30-fold) derives from the use of 2% (w/v) polyvinylpyrrolidone (average Mr 40,000) instead of bovine serum albumin or denatured hemoglobin as the quenching agent (or carrier) during incubation with 125I-wheat germ agglutinin in detergent-free, phosphate-buffered saline. Under these conditions, specific labeling with 125I-wheat germ agglutinin is observed for orosomucoid derivatives that display N-acetylglucosamine or sialic acid residues at the nonreducing termini of their oligosaccharides, as well as for a number of glycoprotein components of a rat hepatocyte plasma membrane fraction. An additional improvement in sensitivity (up to 10-fold) results from an increase in the binding of 125I-wheat germ agglutinin to sialic acid-containing glycoproteins after treatment of the blots with 5 mM sodium metaperiodate followed by 5 mM aniline in the presence of 30 mM sodium cyanoborohydride. This treatment appears to cause the sequential oxidation and reductive phenylamination of the side chain of glycoprotein sialic acid residues.     

The NADP-dependent trifunctional methylenetetrahydrofolate dehydrogenase purified from mouse liver is immunologically distinct from the mouse NAD-dependent [corrected] bifunctional enzyme

Methylenetetrahydrofolate dehydrogenase - methenyltetrahydrofolate cyclohydrolase - formyltetrahydrofolate synthetase was purified to homogeneity from mouse liver, taking advantage of its very high affinity for 2',5'-ADP-Sepharose. Antibodies raised to this trifunctional enzyme and to the bifunctional NAD-dependent dehydrogenase-cyclohydrolase from mouse Ehrlich ascites tumour cells were found not to cross-react with the purified proteins on Western blots. Each of these polyclonal antibodies detects the appropriate protein in extracts of Ehrlich ascites tumour cells after sodium dodecyl sulfate - polyacrylamide gel electrophoresis and electrophoretic transfer of the proteins to nitrocellulose. The procedure has also been used to obtain a purified preparation of the trifunctional enzyme from human liver obtained at autopsy.     

Western blotting and immunochemical detection of histones electrophoretically resolved on acid-urea-Triton- and sodium dodecyl sulfate-polyacrylamide gels

We have developed a method for the efficient transfer of histones from acetic acid-urea-Triton X-100 (AUT)-polyacrylamide minislab gels to nitrocellulose. The AUT gel was equilibrated with 50 mM acetic acid and 0.5% sodium dodecyl sulfate and then with 62.5 mM Tris-HCl, pH 6.8, and 2.3% sodium dodecyl sulfate. An alkaline transfer buffer [25 mM 3-(cyclohexylamino)-1-propanesulfonic acid, pH 10, with 20% methanol] was used to electrophoretically transfer the strongly basic proteins from AUT or sodium dodecyl sulfate gels to nitrocellulose. The applicability of this approach in the immunochemical detection of ubiquitinated histone species is demonstrated.     

Simultaneous quantification of sialyloligosaccharides from human milk by capillary electrophoresis

The acidic oligosaccharides of human milk are predominantly sialyloligosaccharides. Pathogens that bind sialic acid-containing glycans on their host mucosal surfaces may be inhibited by human milk sialyloligosaccharides, but testing this hypothesis requires their reliable quantification in milk. Sialyloligosaccharides have been quantified by anion exchange high-performance liquid chromatography (HPLC), reverse- or normal-phase HPLC, and capillary electrophoresis (CE) of fluorescent derivatives; in milk, these oligosaccharides have been analyzed by high pH anion exchange chromatography with pulsed amperometric detection and, in our laboratory, by CE with detection at 205nm. The novel method described here uses a running buffer of aqueous 200mM NaH2PO4 (pH 7.05) containing 100mM sodium dodecyl sulfate (SDS) mixed with 45% (v/v) methanol to baseline resolve 5 oligosaccharides and separate all 12. This allows automated simultaneous quantification of the 12 major sialyloligosaccharides of human milk in a single 35-min run. This method revealed differences in sialyloligosaccharide concentrations between less and more mature milk from the same donors. Individual donors also varied in expression of sialyloligosaccharides in their milk. Thus, the facile quantification of sialyloligosaccharides by this method is suitable for measuring variation in expression of specific sialyloligosaccharides in milk and their relationship to decreased risk of specific diseases in infants.     

Monoclonal antibodies block cell-cell adhesion in Dictyostelium discoideum

Of 39 monoclonal antibodies that bind the cell surface of aggregating Dictyostelium discoideum, 4 block 76-98% of cell-cell adhesion measured in an in vitro assay. The active antibodies all bind in the range of 10(6) antigenic sites/cell surface and react with more than one material on nitrocellulose blots prepared after polyacrylamide gel electrophoresis of whole aggregating cells in sodium dodecyl sulfate. Active antibodies can by grouped into two classes, each with two very similar members. Class I binds several molecules that are prominent in aggregating cells but scarce or undetectable in vegetative cells, blocks cell adhesion only in the presence of EDTA, and has no detectable effect on cell morphology. Class II binds a wide range of molecules present in both vegetative and aggregating cells, inhibits adhesion as well in the absence as in the presence of EDTA, and reversibly alters cell shape.     

Calmodulin-binding proteins: visualization by 125I-calmodulin overlay on blots quenched with Tween 20 or bovine serum albumin and poly(ethylene oxide)

To streamline detection of calmodulin-binding proteins, blotting techniques for the electrophoretic transfer of proteins onto nitrocellulose filters, followed by overlay with 125I-calmodulin, have been adapted. Autoradiography of the 125I-calmodulin-labeled blots allows the identification and quantitation of proteins that possess affinity for calmodulin. Five protocols for suppressing nonspecific binding and for enhancing specific interactions of 125I-calmodulin with electrophoretically separated proteins were investigated. Tween 20 and bovine serum albumin alone, as well as combinations of bovine serum albumin and poly(ethylene oxide) or hemoglobin and gelatin, were evaluated as quenching and enhancing agents. Tween 20 proved highly effective for quenching nonspecific binding and for enhancing specific 125I-calmodulin binding of a 61,000-Mr rat brain protein, which was only faintly observed on blots quenched with proteins alone. However, Tween 20 dissociated 50% of 68,000-Mr proteins and 80% of 21,000-Mr 125I-labeled protein standards from the nitrocellulose filter. An alternative, the combination of bovine serum albumin followed by incubation with 15,000- to 20,000-Mr poly(ethylene oxide), proved satisfactory for the recovery of 61,000-Mr calmodulin-binding activity and for the detection of calmodulin-binding peptides (50,000 to 14,000 Mr) produced by limited proteolysis of rat brain 51,000-Mr calmodulin-binding protein. These blotting procedures for detection of calmodulin-binding proteins are compatible with a variety of one-dimensional and two-dimensional electrophoresis systems, including a two-dimensional electrophoresis system utilizing urea and sodium dodecyl sulfate in the first dimension and nonurea sodium dodecyl sulfate electrophoresis in the second, a system which proved useful for resolving calmodulin-binding proteins displaying anomalous electrophoretic migration in the presence of urea.     

Immobilization and characterization of cellulase on concanavalin A (Con A)-layered calcium alginate beads

Cellulase has been immobilized on hybrid concanavalin A (Con A)-layered calcium alginate–starch beads. Immobilized cellulase retained about 82% of its activity. Con A was extracted from jack bean and the obtained crude protein was characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis. The immobilized beads showed high mechanical and storage stability; immobilized cellulase retained 100% and 85% activity at 4°C and 30°C, respectively, over one month. The immobilized cellulase retained about 70% of its activity after five cycles of use. The immobilized cellulase retained 70% activity after 120-min exposure to 60°C, whereas the soluble form only retained about 20%, showing that immobilization improved thermal stability. Surface morphology and elemental analysis of immobilized cellulase were examined using scanning electron microscope equipped with energy-dispersive X-ray. Based on the enzyme stability and reuse, this method of immobilization is both convenient and cheap.     

Saccharomyces cerevisiae chromatin associated protein Yp20 is a guanine nucleotide binding protein

Yp20 is a 20kD protein whose role is still obscure which copurifies with yeast histones. Yeast histones were separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose. Incubation of the nitrocellulose blots with [gamma-35S] GTP gamma S demonstrated that Yp20 is a GTP binding protein. A polyclonal antiserum raised against purified Yp20 cross reacted with bacterially expressed cHras and T24 Hras genuine ras products. The results obtained suggest that Yp20 is a yeast chromatin associated ras-related antigen.     

Fluorescent staining of proteins transferred to nitrocellulose allowing for subsequent probing with antisera

A sensitive staining method for protein blots on nitrocellulose is described. It is based on the coupling of a fluorochrome, dichlorotriazynylaminofluorescein, to protein which yields products colorless in visible light but colored when protein blots are illuminated with long-range ultraviolet light. The coupling of a fluorochrome does not affect the antigenic properties of proteins and the stained blots can be subsequently probed with antisera. Thus, the method allows for the unambiguous identification of antigenic proteins transferred to nitrocellulose from sodium dodecyl sulfate-polyacrylamide gels.     

Rapid affinity-purification and biotinylation of antibodies.

A simple and rapid method to affinity-purify and biotinylate antibodies was developed. The method utilizes separation of antigens by sodium dodecyl sulfate polyacrylamide gel electrophoresis, followed by transfer to nitrocellulose and binding of the antibodies to the specific antigen. The antibodies are biotinylated, while still bound to the antigen, thus avoiding the conjugation of the active antigen-binding sites of the antibodies. These antibodies have been successfully used in double-label immunofluorescence studies, but they should be likewise applicable in other immunological protocols.     

Stabilizing hyperactivated lecitase structures through physical treatment with ionic polymers

Lecitase Ultra has been covalently immobilized on cyanogen bromide cross-linked 4% agarose (CNBr) beads, maintaining 70% of the initial activity. The activity of the immobilized enzyme was improved in the presence of Triton X-100, sodium dodecyl sulfate (SDS), and cetyltrimethyl ammonium bromide (CTAB) (e.g., up to 800% when using CTAB). However, CTAB and Triton X-100 presented a negative effect on enzyme stability even at low concentrations, and SDS cannot be used for a long time at 1% concentration. To maintain the hyperactivated conformation of the enzyme in the absence of detergent, ionic polymers were added during incubation of the immobilized enzyme in the presence of detergents. Coating the immobilized enzyme with polyethylenimine in aqueous buffer (PEI) produced a 3-fold increase in enzyme activity. However, in the presence of 0.1% SDS (v/v), this coating produced a 50-fold increase in enzyme activity. Using PEI and 0.01% (v/v) CTAB, the Lecitase activity decreased to 10%. Using irreversible inhibitors, it could be shown that the PEI/SDS-CNBr-Lecitase preparation allowed its catalytic Ser to be more accessible to the reaction medium than the unmodified CNBr-Lecitase.     

Limited modifications of soya proteins by immobilized subtilisin: comparison of products from different reactor types

A comparison of different immobilized enzyme reactors has been made for the limited modification of soya storage proteins and the products compared with those from action of the soluble enzyme. Clarified total water extracts of soya protein were subjected to the action of subtilisin in a soluble and immobilized form. The sodium dodecyl sulfate (SDS) electrophoresis patterns of soya proteins modified by enzyme in the two forms differed for unbuffered soya protein at the same pH of 8.0. However, identical patterns could be obtained by a downward adjustment of the pH of soya protein treated with immobilized enzyme. The same SDS electrophoresis pattern could be obtained for a packed column of immobilized enzyme and a well-mixed vessel by buffering. Operation of the column reactor at higher superficial linear velocities (above 1.47 cm/min), higher protein concentrations (8.8% w/v), and prolonged periods (24 h) led to a bed compression attributed to the protein coating of the support.     

胶束电动色谱法分析奶中两种主要的共轭亚油酸异构体

以胶束电动色谱法对奶样中共轭亚油酸主要的两种异构体进行了分析。在优化条件下(80 mM pH9.0的磷酸盐缓冲液,54 mMSDS,4%(w/v)β-CD,8 M尿素,4%(v/v)乙醇作为运行缓冲液,分离电压25 kV,柱温20℃),胶束电动色谱可在15 min内对奶样中两种主要CLA,即9c,11t-CLA和10t,12c-CLA进行分离测定,最低检出限为0.081 ng/mL。分析结果显示,不同处理奶样中的CLA含量差异显著(P<0.001),但CLA的组成相近,其中的10t,12c-CLA含量差异不显著P=0.999,约为3%;不同品种奶样,如牛奶、水牛奶和羊奶中的CLA含量差异显著(P<0.001),其中CLA含量次序为牛奶>羊奶>水牛奶,并且不同品种奶的9c,11t-CLA与10t,12c-CLA比例差异显著(P<0.05)。     

Elimination of artifacts on native Western blots arising from endogenous lectin activity

While studying the behavior of profilin from Phaseolus vulgaris seeds under native conditions, a high molecular weight species suggesting a complex of profilin and associated proteins was observed by Western immunoblotting. This putative complex was also observed when enzyme-linked secondary antibodies alone were used, and this apparently resulted from antibody association, through its glycosyl moieties, with the endogenous carbohydrate-binding activity from the seed extracts. This endogenous activity corresponded to that of purified phytohemagglutinin (PHA). In addition, the P. vulgaris lectin activity was very stable and was observed when the extracts were pretreated with varying concentrations of sodium dodecyl sulfate, Triton X-100, urea and β-mercaptoethanol, or when membrane blots were boiled in water before incubation with antibody. The activity was abolished only if the membrane was boiled in 1% sodium dodecyl sulfate. This finding could also be useful to implement assays for carbohydrate-binding activity from cell or tissue extracts using different visualizable reagents bearing particular glycosyl moieties.