Sequential sodium dodecyl sulfate-polyacrylamide gel electrophoresis and reversed-phase chromatography of unfolded proteins

Sequential sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis and reversed-phase high performance liquid chromatography on a fluorocarbon packing (poly F column, DuPont) provide fully denatured but highly purified protein, which is free of low-molecular-weight substances and directly amenable to structural analysis. Conditions for the preparative elution of four test proteins (bovine serum albumin, carbonic anhydrase, myoglobin, cytochrome c) blotted to Immobilon membranes have been optimized. Phenol saturated with 50 mM Tris-HCl (pH 8.25) and containing 2% SDS and 1% 2-mercaptoethanol is able to elute proteins that have been blotted to Immobilon membranes, stained with Coomassie R-250, and stored as dry sheets, largely irrespective of their molecular mass. Polypeptides that are not degraded by exposure to low pH can also be efficiently extracted directly from stained gels with 70% formic acid. If further separation of polypeptides is not needed, a simple run of a protein sample dissolved in 1-2% SDS on the poly F column will efficiently remove low-molecular-weight substances, including SDS.     

Structure of the cytochrome c oxidase complex of rat liver. 2. Topological orientation of polypeptides in the membrane as studied by proteolytic digestion and immunoblotting

The orientation of the thirteen polypeptides of rat-liver cytochrome c oxidase in the inner mitochondrial membrane was studied by proteolytic digestion of mitoplasts and sonicated particles. After separation by sodium dodecylsulfate gel electrophoresis proteins were transferred on nitrocellulose, and individual polypeptides were identified by incubation with polypeptide-specific antisera, followed by fluorescein-isothiocyanate-conjugated protein A. The three catalytic polypeptides I-III and seven nuclear coded polypeptides (IV, Vb, VIa, VIc, VIIa, VIIb and VIII) were found accessible to proteases from the cytoplasmic phase. Polypeptides II, IV, Va, Vb and VIa were accessible from the matrix phase, indicating a transmembraneous orientation of polypeptides II, IV, Vb and VIa. Together with data on cross-linking and on cytochrome-c-protected labeling of polypeptides, a model of the cytochrome c oxidase complex was developed. It is suggested that the cytochrome c binding site on polypeptide II is surrounded by several nuclear-coded polypeptides, which may modulate the affinity of the enzyme towards cytochrome c.     

Two-dimensional protein mapping by gold stain and immunoblotting

A method is described for mapping individual polypeptides on a two-dimensional separation of a complex mixture of proteins. Polypeptides are identified by immunoblotting on nitrocellulose prestained for total protein by a colloidal gold stain which does not inhibit antibody binding. The same polypeptides can subsequently be identified on gold-stained blots run under identical conditions, without the need for immunoblotting.     

Ionophore properties of peptide fragments of brain Na+,K+-Atpase

Polypeptide fragments from Na+, K+-ATPase of cattle brain are obtained by the bromocyan-treatment of the protein and subsequent gel filtration via sephadexes; the fragments were reconstructed into bilayer lipid membranes (BLM). Polypeptides of fractions I and II induce cationic and polypeptides of fraction IV--cationic-anionic conductivity of BLM. Neither sodium nor potassium selectivity of BLM modified by protein fragments of fractions I and II was observed. Fluctuations of the modified membranes current are of spasmodic character, ATP and inhibitors of the sodium pump do not affect them. The induction of current fluctuations peculiar to channels into BLM is supposed to be a character of polypeptides obtained after the ATPase splitting but not of the cation-transport system of the sodium pump.     

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.     

Isolation from human erythrocytes of a new membrane protein which inhibits the formation of complement transmembrane channels

A protein which inhibited complement channel formation was isolated from extracts of papain-digested human erythrocyte membranes using DEAE-Sephacel, Bio-Gel A0.5m column chromatographies, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by transfer to nitrocellulose paper and elution with 2% NP-40 solution. The purified protein showed a molecular weight of 18 kDa, and efficiently inhibited hemolysis of EC5-7 cells with C8 and C9, but did not show any decay-accelerating activity to C5 convertase. Immunochemical analysis of native membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis using the antibody against this protein gave a single band having the same mobility as this protein; papain did not eliminate a significant portion of this protein.     

The erasable Western blot

A method for successfully removing primary and secondary antibodies from nitrocellulose blots while preserving the originally immobilized polypeptides was developed. Polypeptides were separated by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and electrophoretically transferred to nitrocellulose. Nonspecific binding sites were blocked with 5% (w/v) nonfat dried milk. After blots were reacted sequentially with antibodies directed against the antigen of interest and with radiolabeled secondary antibody, a 10-min wash in 5% (w/v) milk was required prior to drying and autoradiography. A 30-min incubation at 70 degrees C in 2% (w/v) sodium dodecyl sulfate containing 100 mM beta-mercaptoethanol quantitatively removed the antibodies and allowed reuse of the blot. A modification of this method similarly allowed reuse of Western blots when proteins were immobilized on nylon. Potential applications and limitations of this method are discussed.     

Protein organization of rat synaptic plasma membranes and synaptic vesicles: A one- and two-dimensional study

The protein organization of rat brain synaptic plasma membranes (SPM) and synaptic vesicles (SV) was investigated by surface iodination and one- and two-dimensional electrophoresis. Polypeptides of molecular weights (MWs, in Kilodaltons) 170 K, 135 K, 96-86 K, 68-64-61 K, 56 K, 52 K, 38 K, 35-33 K, and 18 K are predominantly or exclusively exposed on the extracellular side of synaptosomes. Several polypeptides of MW between 70 K and 40 K are exclusively exposed on the cytoplasmic side of SPM. The use of two-dimensional electrophoresis allowed to recognize that, for some classes of MW, there are polypeptides of nearly the same MW and different isoelectric points exposed on both sides of SPM. The synaptosomal membrane shows a predominance of acidic proteins on the extracellular side and more neutral and basic proteins on the cytoplasmic side. With respect to SPM, SV are particularly enriched with polypeptides of MW 71 K, 56 K, 39-38 K, 32 K, 16 K, and 15 K. One of them, a doublet of MW 39-38 K, is the most highly labeled species upon surface iodination and is similar, but not identical, with a doublet located on the cytoplasmic side of SPM.     

Sub-set characteristics of DNA sequences involved in tight DNA/polypeptide complexes and their homology to nuclear matrix DNA

Polypeptides co-isolating with DNA induce the binding of a fraction of native DNA fragments to nitrocellulose filters. Southern analysis reveals a high intensity of self-hybridization of the DNA sequences retained on nitrocellulose filters. Consistently, the DNA fraction passing the filters shows only weak hybridization when probed with DNA retained on filters. This indicates that the DNA/polypeptide complexes reside on a non-random sub-set of DNA sequences. Moreover, a high degree of homology was found between residual nuclear matrix DNA sequences and the DNA sequences retained on nitrocellulose filters. This indicates that the DNA sequences associated with tightly bound polypeptides originate from sites where the genome is salt-stably anchored in the nuclear matrix.     

Preparative elution of proteins from nitrocellulose membranes after separation by sodium dodecyl sulfate-polyacrylamide gel electrophoresis

Various conditions were analyzed and optimized for the preparative elution of proteins from nitrocellulose membranes after transfer from sodium dodecyl sulfate (SDS)-polyacrylamide gels. The efficiency of elution was best using pyridine or acetonitrile elution solvents, intermediate for buffer containing a mixture of sodium dodecyl sulfate, Triton X-100, and sodium deoxycholate, and negligible for buffers containing any single detergent or chaotropic salt, such as urea or guanidine hydrochloride. The efficiency of elution with any solvent also depended on the molecular weight of the proteins, smaller proteins being more easily removed from membranes. As a general procedure, proteins may be eluted from nitrocellulose membranes by incubation with either 40% acetonitrile or 50% pyridine in 0.1 M ammonium acetate, pH 8.9, for 1-3 h at 5-37 degrees C. The recommended procedures for protein elution appear to offer a rapid, simple, and efficient means of recovering proteins from complex mixtures after separation by SDS-PAGE and transfer to nitrocellulose membranes.