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.     

Efficient transfer of proteins from acetic acid-urea and isoelectric-focusing gels to nitrocellulose membrane filters with retention of protein antigenicity

A method which facilitates the rapid and quantitative electrophoretic transfer of proteins from gels not containing sodium dodecyl sulfate (SDS) to nitrocellulose membranes is described. The equilibration of non-SDS-polyacrylamide gel electrophoretic gels in a buffer containing SDS confers a net negative charge to the proteins present, presumably as a result of the formation of SDS-protein complexes. Proteins from gels equilibrated in the SDS buffer and then electroblotted in a Tris-glycine buffer at pH 8.3 are transferred with much greater efficiency than are proteins from untreated gels. The method has been shown to significantly enhance the electrophoretic transfer of polyoma viral proteins resolved in either acetic acid-urea or isoelectric-focusing gels to nitrocellulose membranes, and it is suggested that the method should have universal applicability to all gel electrophoresis systems currently employed. The proteins from isoelectric-focusing gels treated with SDS and transferred to nitrocellulose membranes were found to retain antigenicity to antisera prepared against either denatured or native viral proteins.     

A two-step procedure for efficient electrotransfer of both high-molecular-weight (greater than 400,000) and low-molecular-weight (less than 20,000) proteins

We have developed conditions for the efficient electrotransfer from polyacrylamide gels to nitrocellulose sheets of a broad size range of proteins (Mr 8,000 to Mr greater than 400,000). The important features of this procedure include a two-step electrotransfer, beginning with elution of low-molecular-weight polypeptides at a low current density (approximately 1 mA/cm2) for 1 h, followed by prolonged electrotransfer (16-20 h) at high current density (approximately 3.5-7.5 mA/cm2) in conditions that favor the elution of high-molecular-weight proteins. The transfer buffer includes 0.01% sodium dodecyl sulfate to enhance protein elution, and 20% methanol to improve the retention of proteins on the nitrocellulose sheet. The nitrocellulose is air-dried after transfer is complete to eliminate loss of proteins during subsequent processing. This transfer procedure works well with proteins prepared from many different cell types, and is suitable for use with all polyacrylamide gel systems tested. With little or no modification, our method should also be applicable to transfer membranes other than nitrocellulose.     

A silver-staining technique for detecting minute quantities of proteins on nitrocellulose paper: retention of antigenicity of stained proteins

A method using a silver-staining procedure to detect minute quantities of proteins on nitrocellulose paper is described. The technique is sensitive enough to detect nanogram quantities of proteins resolved on sodium dodecyl sulfate-polyacrylamide gels and then transferred to nitrocellulose paper by the electrotransfer technique. After the staining procedures, the proteins are shown to retain their antigenic properties.     

Tips on improving the efficiency of electrotransfer of target proteins from Phos‐tag SDS‐PAGE gel

The sensitivity of Western blotting analysis after Phos‐tag SDS‐PAGE is occasionally inferior to that after normal (Phos‐tag‐free) SDS‐PAGE under similar experimental conditions, possibly as a result of inefficient electrotransfer from the Phos‐tag gel to the blotting membrane. We therefore present tips on improving the efficiency of electrotransfer of proteins in semidry and wet‐tank blotting. When model samples containing several standard phosphoproteins were subjected to semidry blotting, their electrotransfer efficiencies after Phos‐tag SDS‐PAGE were markedly inferior to those of their dephosphorylated counterparts in the same gel. This was ameliorated by immersing the electrophoresed Phos‐tag gel in a transfer buffer containing 1 mM EDTA for 30 min before electroblotting. Similarly, phosphoproteomes in crude cell extracts were inefficiently transferred by semidry blotting, but the efficiencies of their electrotransfer were improved by pretreatment with EDTA. In contrast, the efficiencies of wet‐tank blotting of the same samples were not dependent on the degree of phosphorylation, and the efficiencies of electrotransfer of all proteins from Phos‐tag gels were similar to those from normal gels. In some cases involving the use of a Phos‐tag gel, addition of 0.1% w/v of SDS to the transfer buffer significantly improved the electrotransfer.     

Immunoblotting from immobilized pH gradients. The case of alpha 2-macroglobulin

Guidelines for effective blotting of proteins from immobilized pH gradients with a soft polyacrylamide matrix (e.g. T% = 3) include: thick (1 mm) IPG slabs, electrotransfer in a buffer tank in the presence of 0.1% SDS, nitrocellulose of the sturdiest type, thorough removal of all IPG fragments before further processing of the membrane. For alpha 2-M, IPG on a 4-6.5 gradient followed by enzyme-linked immunodetection allows the recognition of a complex pattern with several bands centered around pI 5.1. The procedure may also reveal the desialylated forms of alpha 2-M (microheterogeneity reduced to 2-3 bands), the native subunits (after reduction with thiols) and the denatured half molecules (in the presence of 8 M urea).     

Visualization of acid phosphatase activity on nitrocellulose filters following electroblotting of polyacrylamide gels

A method for visualizing acid phosphatase isoenzymes by activity staining on nitrocellulose filters after electroblotting of proteins fractionated on nondenaturing polyacrylamide gels is described. Reproducible results were obtained when 25 mM Tris-192 mM glycine was used as the transfer buffer instead of 0.7% acetic acid, 50 mM sodium acetate, pH 4, or 0.14 M acetic acid--0.35 M beta-alanine, pH 4.3. Dot-blot analysis of banana fruit extracts on nitrocellulose filters revealed that a minimum of 5 x 10(-3) units (nmol p-nitrophenyl phosphate hydrolyzed g-1.h-1) of acid phosphatase activity can be detected. This method can be suitable for screening a large number of biological samples for monitoring acid phosphatase activity.     

A procedure for the immunoblotting of proteins separated on isoelectric focusing gels

A method has been devised for performing Western blot assays on proteins resolved by isoelectric focusing. Electrophoretic transfer of proteins directly from isoelectric focusing (IEF) tube gels to nitrocellulose sheets allowed their immunoassay without conventional second dimension SDS gel electrophoresis. The same method can also be used for IEF slab gels. For the immunostaining of nonmuscle actin isoforms in extracts of cultured cells, the resolution of this technique was much improved over that of Western blots of two-dimensional gels.     

Staining of proteins on SDS polyacrylamide gels and on nitrocellulose membranes by Alta, a colour used as a cosmetic

We describe here the use of Alta, a pre-existing scarlet-red stain of cosmetic use, for staining proteins on sodium dodecyl sulfate (SDS) polyacrylamide gels, as well as for a single step staining of gels and nitrocellulose membranes during Western blot analysis. This stain, which is composed of 0.8% Crocein scarlet (brilliant crocein) and 0.2% Rhodamine B, is inexpensive, easy to use and nearly as sensitive as Coomassie Brilliant Blue (CBB) R-250. The gels can be stained in 10% Alta (2 h) and can be destained effectively only with 7% acetic acid as opposed to the conventional destainer (methanol/acetic acid/water) required for CBB-stained gels. In an alternative procedure, the proteins can be stained on the gel while electrophoresis by simply using 5% Alta in the top tank buffer and the stain can be viewed under UV-transilluminator. This procedure can also be used for Western blot analysis, as a single step procedure for staining of proteins on the gel as well as on the nitrocellulose membrane, as the stain is retained on the membrane after protein transfer. Thus, this staining procedure allows monitoring of proteins after each step in the Western blot, thereby eliminating the need to run separate gels for staining and Western blot analysis, and also the need for Ponceau Red S staining of the nitrocellulose membrane during Western blot analysis.     

Microheterogeneity of Micro tubule-Associated τ Proteins Is Due to Differences in Phosphorylation

We have studied the heterogeneity of the microtubule-associated tau proteins using tau-specific antibodies and two-dimensional electrophoresis. Both monoclonal and polyclonal antibodies to tau proteins recognize five bands in cow brain microtubule proteins run on sodium dodecyl sulfate (SDS)-polyacrylamide gels, with apparent molecular weights between 56,000 and 66,000. Immunoblots of cow brain microtubules separated on two-dimensional gels, using nonequilibrium pH gradient electrophoresis in the first dimension and SDS-gel electrophoresis in the second, reveal that greater than 30 isoforms of tau exist. The tau proteins vary in pI from 6.5 to 8.5, with the higher-molecular-weight forms being more acidic. The microheterogeneity of tau is not induced by cycling of microtubules, because two-dimensional immunoblots of tau from total brain are almost identical to those of tau from cycled tubules. Adult rat brain tau, which appears as three doublet bands on SDS gels, also exhibits considerable isoelectric heterogeneity, as does tau from 7-day-old rats, which appears as only one band on SDS gels. After dephosphorylation of cow brain tau with alkaline phosphatase, the highest-molecular-weight band disappears on SDS gels. On two-dimensional gels, the number of tau variants decreases by more than half after dephosphorylation, and the more basic species increase greatly in intensity. Preliminary experiments with tau labeled in vivo with 32PO4 also indicate that the more acidic tau proteins are the more highly phosphorylated forms. Thus, isoelectric heterogeneity of tau proteins exists at all ages and is due, at least in part to differences in the state of phosphorylation of tau isoforms.     

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.     

Induction of 33-kD and 60-kD Peroxidases during Ethylene-Induced Senescence of Cucumber Cotyledons

Ethylene enhanced the senescence of cucumber (Cucumis sativus L. cv `Poinsett 76') cotyledons. The effect of 10 microliters per liter ethylene was inhibited by 1 millimolar silver thiosulfate, an inhibitor of ethylene action. An increase in proteins with molecular weights of 33 to 30 kilodaltons and lower molecular weights (25, 23, 20, 16, 12, and 10 kilodaltons) were observed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels after ethylene enhanced senescence. The measurement of DNase and RNase activity in gels indicated that these new proteins were not nucleases. Two proteins from ethylene-treated cotyledons were purified on the basis of their association with a red chromaphore and subsequently were identified as peroxidases. The molecular weights and isoelectric points (pI) of two of these peroxidases were 33 kilodaltons (cationic, pI = 8.9) and 60 kilodaltons (anionic, pI = 4.0). The observation that [³⁵S]Na₂SO₄ was incorporated into these proteins during ethylene-enhanced senescence suggests that these peroxidases represent newly synthesized proteins. Antibodies to the 33-kilodalton peroxidase precipitated two in vitro translation products from RNA isolated from ethylene-treated but not from control cucumber seedlings. This indicates that the increase in 33-kilodalton peroxidase activity represents de novo protein synthesis. Both forms of peroxidase degraded chlorophyll in vitro, which is consistent with the hypothesis that peroxidases have catabolic or scavenging functions in senescent tissues.     

Microsequence analysis of peptides and proteins. VIII. Improved electroblotting of proteins onto membranes and derivatized glass-fiber sheets

We have quantitatively examined the various parameters affecting the electrotransfer and sequence analysis of proteins from sodium dodecyl sulfate (SDS) gels to derivatized glass fiber paper or to polyvinyldifluoride (PVDF) membranes. Transfer yields in the range of 90-95% can be obtained for proteins in the molecular weight range of 10-90 kDa for transfer from 12% SDS gels to glass fiber paper derivatized with either QAPS (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) or APS (aminopropyltriethoxysilane). In order to achieve these yields, it was necessary to modify the conditions described by R. Aebersold et al. (J. Biol. Chem. 261, 4229-4238, 1986). We activated the glass fiber paper with dilute ammonia water and derivatized the activated glass fiber paper with QAPS and APS in anhydrous solvents which were allowed to slowly absorb moisture during the derivatization process. The transfer yield varied with transfer time versus molecular weight of the protein for a given percentage gel. Shorter transfer times and higher yields were obtained for higher molecular weight proteins on 8% gels. Lower molecular weight protein gave higher yields from 12% gels under similar transfer conditions. Sequencing yields of the transferred proteins were in the range of 40-80%, but a number of background peaks were observed on HPLC analysis of the phenylthiohydantoin amino acid derivatives. Transfer yields in the range of 85-95% were observed for similar experiments with PVDF membranes. In order to achieve these yields, it was necessary to modify the conditions described by P. Matsudaira (J. Biol. Chem. 262, 10035-10038, 1987). A lower voltage and longer transfer times gave higher transfer yields. In order to achieve consistently high transfer yields, it was also necessary to precoat the PVDF membranes with Polybrene. The PVDF membranes were cut into approximately 1-mm-wide strips and inserted into a continuous flow reactor (J. E. Shively, P. Miller, and M. Ronk, Anal. Biochem. 163, 517-525, 1987) for sequence analysis. Overall yields of samples loaded onto gels, electrotransferred to Polybrene-coated PVDF membranes, and sequenced ranged from 50-60% for beta-lactoglobin (10-50 pmol loaded onto SDS gels) to 20-30% for bovine serum albumin and soybean trypsin inhibitor (50 pmol loaded onto SDS gels). A comparison of the two methods shows clear advantages for the PVDF membranes over the derivatized glass fiber paper, including the ability to directly sequence the Coomassie blue-stained PVDF membranes, and the lower backgrounds observed on subsequent sequence analysis.     

Detection and Characterization of Some New Basic Proteins in Chicken Postsynaptic Densities

Chicken brain postsynaptic density (PSD) polypeptides, obtained by treating synaptosomes with 0.5% Triton X-100 and then further purified on a sucrose gradient, are demonstrated to contain four basic proteins of 76K (pI greater than 9.2), 58K (pI 8.1-8.8, heterogeneous), 40K (pI 9.0), and 24K (pI 8.9). Nonequilibrium pH gradient-sodium dodecyl sulfate two-dimensional gels further reveal six more basic proteins with pI values higher than 9.2: 76K, 52K, 47K, 45K, 36K, and 34K. These basic proteins are a major part of the total chicken PSD polypeptides appearing on the gels. Some of these basic proteins (58K, 52K, 47K, 36K, 24K, and two at 76K) are distinguishable from those of brain mitochondria, the major contaminant. The 40K and 34K proteins may be common mitochondrial polypeptides. The 45K protein is probably a mitochondrial contaminant. A number of proteins including 76K (synapsin I-like protein) and 58K, along with some other minor ones, can be phosphorylated by endogenous protein kinase(s) in the presence of Ca2+, Mg2+, and [gamma-32P]ATP. No PSD basic proteins bind Ca2+.     

Sodium dodecyl sulfate-mediated transfer of electrophoretically separated DNA-binding proteins

A modified procedure for the transfer of electrophoretically-separated proteins from sodium dodecyl sulfate (SDS)-polyacrylamide gels onto nitrocellulose filters has been developed. During the diffusion mediated transfer, the SDS-protein complexes were maintained and SDS was added to the buffer. This increases the number of polypeptide species bound to the filter thereby giving an accurate replica of the original gel pattern. The immobilization in the gel of certain polypeptides characterized as DNA-binding proteins, which is observed when SDS is eliminated prior to blotting is avoided. The molecules blotted in the presence of SDS remain immunoreactive and able to bind DNA.     

Fluorescent labeling of cysteinyl residues to facilitate electrophoretic isolation of proteins suitable for amino-terminal sequence analysis

A protein labeling procedure which enables detection of subpicomole quantities of proteins on sodium dodecyl sulfate (SDS)-polyacrylamide gels is described. Proteins are rendered fluorescent by reduction of disulfide bonds with dithiothreitol followed by alkylation with 5-N-[(iodoacetamidoethyl)amino]naphthalene-1-sulfonic acid (5-I-AEDANS) or 5-iodoacetamido-fluorescein. Labeling is performed prior to electrophoresis, thus eliminating the need for staining with dyes and destaining after electrophoresis. As little as 375 fmol (25 ng) of prelabeled bovine serum albumin can be readily visualized after electrophoresis. Bands are still visible after electrophoretic transfer to nitrocellulose. Simultaneous labeling of proteins in complex mixtures is possible using this technique. This includes cysteine containing proteins of disrupted Newcastle disease virus. The magnitudes of the molecular weight increases which occur upon labeling reflect the cysteine contents of proteins. The mode of chemical modification for the prelabeling procedure was chosen because of its compatibility with analytical techniques, such as amino acid analysis, peptide mapping, or sequence analysis, which may be applied to the protein after electroelution from SDS-acrylamide gels. It replaces the need for reduction and carboxymethylation prior to these analytical procedures. Protein-sequence analysis of prelabeled bovine serum albumin, including samples electroeluted from SDS-acrylamide gels, has justified the choice of this method to facilitate isolation of proteins for sequence analysis. Equivalent sequence data were obtained with reduced bovine serum albumin S-alkylated with iodoacetic acid or 5-I-AEDANS.     

Detection of actin-binding proteins in human platelets by 125I-actin overlay of polyacrylamide gels

Actin-binding proteins have been identified in human platelets with a gel-overlay technique that uses 125I-G-actin. Platelet proteins were separated on SDS polyacrylamide gels using the buffer system of Laemmli (1970, Nature [Lond.] 227:680-685). The proteins were fixed in the gels with methanol-acetic acid, the SDS was washed out, and the proteins were renatured. The gels were incubated with 125I-G-actin from rabbit skeletal muscle that was radiolabeled with 125I according to the method of Bolton and Hunter (1973, Biochem. J. 133:529-538) and has been shown to retain biological activity. After nonspecifically bound radioactivity was washed out, gels were dried and processed for autoradiography. The 125I-G-actin binds to several proteins in human platelets, platelet extracts, and the particulate fraction. Control experiments demonstrate that the 125I-G-actin can be displaced by use of increasing amounts of unlabeled actin, that the binding is stable to 0.6 M NaCl, and that preheating the 125I-G-actin to 90 degrees C for 3 min eliminates all binding. Prominent 125I-G-actin-binding activities were present at Mr 90,000 and 40,000. The binding to the 90,000 Mr protein appears to be at least partially Ca++ sensitive, whereas the binding to the 40,000 Mr protein does not. 125I-G-actin bound to proteins in the SDS gels can be fixed in situ and compared directly with the stained gel. This technique should prove generally useful in identification and purification of some actin-binding proteins from cells and tissues.     

A growth factor from spinal cord.

Mitogenic activity is present at a variety of sites in the central nervous system. A growth factor was purified from neonatal bovine spinal cord. It has a pI of 9.5-9.8 and a molecular weight of about 11,000 daltons. Spinal cord growth factor is a basic polypeptide that is inactivated by extremely acid or basic conditions. Its mobility on SDS polyacrylamide gels suggests that this factor is different from pituitary FGF and brain FGF-1.     

Purification and characterization of alpha-amylase from rat pancreatic acinar carcinoma. Comparison with pancreatic alpha-amylase.

alpha-Amylase was purified to apparent homogeneity from normal pancreas and a transplantable pancreatic acinar carcinoma of the rat by affinity chromatography on alpha-glucohydrolase inhibitor (alpha-GHI) bound to aminohexyl-Sepharose 4B. Recovery was 95-100% for both pancreas and tumour alpha-amylases. They were monomeric proteins, with Mr approx. 54000 on SDS/polyacrylamide-gel electrophoresis. Isoelectric focusing of both normal and tumour alpha-amylases resolved each into two major isoenzymes, with pI 8.3 and 8.7. Tumour-derived alpha-amylase contained two additional minor isoenzymes, with pI 7.6 and 6.95 respectively. All four tumour isoenzymes demonstrated amylolytic activity when isoelectric-focused gels were treated with starch and stained with iodine. Two-dimensional electrophoresis, on SDS/10-20%-polyacrylamide-gradient gels after isoelectric focusing, separated each major isoenzyme into doublets of similar Mr values. Pancreatic and tumour-derived alpha-amylases had similar Km and Ki (alpha-GHI) values, but the specific activity of the tumour alpha-amylase was approximately two-thirds that of the normal alpha-amylase. Although amino acid analysis and peptide mapping with the use of CNBr, N-chlorosuccinimide or Staphylococcus aureus V8 proteinase gave comparable profiles for the two alpha-amylases, tryptic-digest fingerprint patterns were different. Antibodies raised against the purified pancreatic alpha-amylase and tumour alpha-amylase respectively showed only one positive band on immunoblotting after gel electrophoresis of crude extracts of rat pancreas and carcinoma, at the same position as that of the purified enzyme. More than 95% of the alpha-amylase activity in the pancreas and in the tumour was absorbed by an excess amount of either antibody, indicating that normal and tumour alpha-amylases are immunologically identical. The presence of additional isoenzymes in the carcinoma, and dissimilarity of tryptic-digest patterns, may reflect an alteration in gene expression or in the post-translational modification of this protein in this heterogeneously differentiated transplantable pancreatic acinar carcinoma.