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.     

Two-dimensional electrophoresis of plasma proteins without denaturing agents.

A technique of two-dimensional polyacrylamide gel electrophoresis for the separation of plasma proteins is described. Human plasma proteins were separated by isoelectric focusing followed by electrophoresis in a 4 to 21% linear gradient gel slab. No denaturing agent was used throughout the procedure, so that the analysis of native proteins is possible. Two-dimensional patterns obtained from normal human plasma samples were recorded as "staining density maps," which are similar to contour line maps, and more than 230 protein spots were counted reproducibly on each "staining density map." This technique permits the simultaneous estimation of pI's and approximate molecular weights of native proteins on the slab gel. Applications of this technique to an IgA myeloma plasma sample and a porcine serum sample are described.     

Apparatus for discontinuous electrophoresis in polyacrylamide gel slabs

Construction of an inexpensive slab discontinuous electrophoresis apparatus is described. Using this apparatus 23 human serum proteins were resolved and the gel could be scanned with a standard densitometer to yield a trace with discrete peaks or pronounced shoulders for each protein band. The advantages of a single homogeneous slab for comparative studies are indicated.     

Recovery of native proteins from preparative electrophoresis gel slices by reverse polarity elution

A technique for high yield recovery of native, biologically active proteins from preparative polyacrylamide gel slices by reverse polarity elution is described. No apparatus other than the standard slab gel electrophoresis system is required. Several proteins have been recovered in biologically active form at a 90% yield, in quantities ranging from 0.4 mg to 4.2 mg. The method is effective with both small (9,000 dalton) and large (186,000 dalton) polypeptides. Both simple and complex proteins are recovered intact. For example, the copper-zinc and manganese superoxide dismutases from crude soybean extracts are active upon recovery. Similarly, the vitamin D-dependent calcium binding proteins from rat kidney and intestine are isolated by this method in homogeneous, active form.     

Anaerobic multiphasic gel electrophoresis of the molybdoproteins in extracts of Clostridium pasteurianum

Simple procedures using multiphasic buffer systems for anaerobic electrophoresis have been devised to identify oxygen-labile metalloproteins. An anaerobic slab gel apparatus was developed with cooling and design for anaerobic conditions. Included is a procedure to remove sample wells after stacking proteins in a crude extract, to prevent streaking (background) caused by continuous leakage of "nonstacked protein" from the sample wells. Identification of eleven Mo zones in extracts of Clostridium pasteurianum demonstrates the usefulness of the technique in identifying radiolabeled oxygen-labile proteins in cell-free crude lysates.     

A versatile apparatus for polyacrylamide and agarose gel electrophoresis in Plexiglas slab gel molds

A simple vertical slab gel electrophoresis apparatus for analytical, preparative, and two-dimensional electrophoresis is described. The use of permanently sealed Plexiglas acrylic plastic slab gel molds which need to be sealed only at the bottom during gel formation, rather than the glass plate sandwich used in most previous designs, virtually eliminates leakage during gel formation and, in addition, permits the continuous monitoring with ultraviolet light of proteins and nucleic acids labeled with fluorescent dyes during electrophoresis. Results obtainable with this apparatus are equivalent to those achieved in other apparati which are more expensive to fabricate or purchase.     

Separation of the two high molecular weight spectrin bands by a newly designed preparative slab electrophoresis technique.

Constructions and operation of an inexpensive preparative slab gel electrophoresis apparatus is described. A slab is made from two wide glass plates with symmetrical windows cut out from both sides. Coating the plates with a silane reagent allows good adhesion of a low concentration acrylamide sodium dodecyl sulfate gel. The migration of extracted chromatographed and fluorescent spectrin mixture is monitored with a uv light. At the end of the run, the slab is turned upside down and the material cluted upward in the small space formed by a dialyzing membrane placed in between the slab and the upper buffer reservoir. Using this apparatus and technique, the two heavy molecular weight spectrin bands can be purified. Advantages of this new system are discussed.     

Modifications and additions to the ISO-DALT system for two-dimensional gel electrophoresis

Modifications of ISO-DALT devices that further enhance the efficiency and reproducibility of two-dimensional mapping of proteins are described. The principal changes in ISO system devices include the introduction of a gel casting trough with a removable panel to permit the removal of excess gel without introducing air into the electrofocusing gels and the introduction of an upper electrode compartment with a separate watertight septum for each electrofocusing tube to permit tube removal for cleaning and replacement. The principal changes in DALT system devices include the use of modified powder funnels to introduce acrylamide solutions into the slab gel gradient former without aeration; the introduction of a flexible outlet system for the gradient former to facilitate the removal of air bubbles; the introduction of an inexpensive two-part mixing chamber to permit disassembly for cleaning; the use of split gel holders to eliminate deformation and breakage of electrofocusing gels during loading onto slab gels; the introduction of an inexpensive integrated slab gel casting/rotating apparatus; and the introduction of a simple, water-cooled slab gel electrophoresis apparatus to reduce the volume of running buffer used in electrophoresis.     

Studies on interaction of 5 S RNA with ribosomal proteins

Proteins of the large ribosomal subunit of rat liver (TP 60) were immobilized by diffusion transfer onto nitrocellulose after two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Incubation of the TP 60 blots with 32P-labeled 5 S RNA under defined ionic conditions (300 mM KCl, 20 mM MgCl2) resulted in specific binding to a limited set of ribosomal proteins consisting of proteins L3, L4, L6, L13/15 and--to a lesser extent--L7 and L19. Under identical conditions, blots with proteins of the small ribosomal subunit (TP 40) did not bind 5 S RNA.     

Comparative analysis of protein aggregates by blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis in a three-dimensional geometry gel

We describe the comparative analysis of protein aggregates by combining blue native electrophoresis and subsequent sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) using a 3-D geometry gel for simultaneous processing of many samples. The first native electrophoresis step, separating the aggregates, is carried out for a series of samples in parallel lanes within a slab gel. This gel is then placed on the top surface of a cylindrical, 3-D geometry gel for the second denaturing electrophoresis step, separating the proteins composing the aggregates. The samples migrate parallel to the vertical axis of the gel cylinder. Data are acquired online by photodetection of laser-induced fluorescence during electrophoresis. For this purpose, the samples are fluorescently labeled within the slab gel after the first separation step. A 3-D geometry gel separates the equivalent of many conventional SDS slab gels represented by vertical layers in the 3-D gel body. In this way, many samples are analyzed in the same gel under identical conditions, improving comparability and resolution and making the process considerably more efficient. This novel technique allowed the identification of several aggregate classes of recombinant proteins expressed in bacteria. We observed that proteins preferentially bind to homolog polypeptides, but also seem to form a trapping mesh co-aggregating with other proteins. The aggregation pattern revealed by this technique supplements data obtained from standard two-dimensional gel electrophoresis analysis. We expect interesting applications, for instance in aggregate monitoring of clinical samples. It should be feasible to quickly gain a diagnostic picture during amyloid-related neurodegenerative disease development or to observe drug effects on protein aggregation.     

An apparatus for thin layer vertical polyacrylamide gel electrophoresis with discontinuous buffer and gel system

A vertical polyacrylamide gel slab electrophoresis apparatus with a discontinuous gel and buffer system and a running gel of 1 mm in thickness was devised. Using this apparatus, which employs stacking and sieving effects, sharp bands comparable to those of disc electrophoresis were obtained.Furthermore, a new application using detection with ultraviolet method was introduced for isozyme study.     

Isoelectric focusing--polynucleotide/polyacrylamide-gel electrophoresis. A technique to separate and characterize nuclease activities.

Individual native nuclease activities from human leucocytes are separated by using two-dimensional gel electrophoresis in an apparatus that allows the simultaneous running of 28 gels. Proteins are separated by isoelectric focusing in a disc gel, followed by electrophoresis into a slab gel containing DNA. Protein denaturants are avoided in the second dimension by the use of a running pH well above the optimal pH for DNAase (deoxyribonuclease) activity. Electrophoresed gels are incubated in appropriate buffers to activate nuclease activity. After staining for intact DNA, the positions of active enzymes, unobscured by the presence of other proteins, are revealed as colourless spots in a reddish-purple field. The technique is easy to use and is sensitive to 50pg of DNAase I. Versatility is provided by the use of either acidic or basic electrophoresis running buffers and by the use of specific gel incubation conditions to reveal different sets of enzyme activities. Two DNAases active at pH 7.4 in the presence of Mg2+ and Ca2+, and sixteen DNAases active at acidic pH and not requiring metals, are detected. Treatment of the human enzymes with specific glycosidases reveals that many of the human DNAases are glycoproteins containing negatively charged moieties and may be derived from modification of parent activities.     

Immunofluorescent localization of 100K coated vesicle proteins

A family of coated vesicle proteins, with molecular weights of approximately 100,000 and designated 100K, has been implicated in both coat assembly and the attachment of clathrin to the vesicle membrane. These proteins were purified from extracts of bovine brain coated vesicles by gel filtration, hydroxylapatite chromatography, and preparative SDS PAGE. Peptide mapping by limited proteolysis indicated that the polypeptides making up the three major 100K bands have distinct amino acid sequences. When four rats were immunized with total 100K protein, each rat responded differently to the different bands, although all four antisera cross-reacted with the 100K proteins of human placental coated vesicles. After affinity purification, two of the antisera were able to detect a 100K band on blots of whole 3T3 cell protein and were used for immunofluorescence, double labeling the cells with either rabbit anti-clathrin or with wheat germ lectin as a Golgi apparatus marker. Both antisera gave staining that was coincident with anti-clathrin, with punctate labeling of the plasma membrane and perinuclear Golgi apparatus labeling. Thus, the 100K proteins are present on endocytic as well as Golgi-derived coated pits and vesicles. The punctate patterns were nearly identical with anti-100K and anti-clathrin, indicating that when vesicles become uncoated, the 100K proteins are removed as well as clathrin. One of the two antisera gave stronger plasma membrane labeling than Golgi apparatus labeling when compared with the anti-clathrin antiserum. The other antiserum gave stronger Golgi apparatus labeling. Although we have as yet no evidence that these two antisera label different proteins on blots of 3T3 cells, they do show differences on blots of bovine brain 100K proteins. This result, although preliminary, raises the possibility that different 100K proteins may be associated with different pathways of membrane traffic.     

Trans-elution: a method for the purification of components of complex biological extracts

Trans-elution is a simple and rapid technique allowing the purification of macromolecules trapped in polyacrylamide gels following electrophoresis. This method of purification consists of the sideways transfer of the macromolecules under the influence of an electric field from the gel slab toward an inert support. In contrast to the role of nitrocellulose used in the "Western blotting" technique, in this case the support does not bind the macromolecules. It consists of a network capable of retaining the buffer by capillary. The electroeluted proteins remain in solution in the buffer and it is thus easy to recover them by spin-drying the support. The support material is either glue-free paper or glass fiber paper. Small concentrated samples are obtained at high yield. The procedure does not require gel slicing, thus avoiding both manipulation of the gel and errors in the localization of the fractions to be purified. In a single step all the proteins fractionated on the gel may be eluted. The trans-elution technique has been applied to the purification of Plasmodium falciparum and Toxoplasma gondii antigens, the causative agents of malaria and toxoplasmosis, respectively.     

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.     

Two-Dimensional Polyacrylamide Gel Electrophoresis of Envelope Proteins of Escherichia coli

A method of separating envelope proteins by two-dimensional polyacrylamide gel electrophoresis is described. Escherichia coli envelopes (inner and outer membranes) were prepared by French pressing and washed by repeated centrifugation. Membrane proteins were solubilized with guanidine thiocyanate and were dialyzed against urea prior to two-dimensional electrophoretic analysis. The slab gel apparatus and conditions were similar to the technique developed by Metz and Bogorad (1974) for the separation of ribosomal proteins. This separation occurs in 8 M urea for the first dimension and in 0.2% sodium dodecyl sulfate for the second dimension. The technique separates about 70 different membrane proteins in a highly reproducible fashion according to both intrinsic charge and molecular weight. Some examples of alterations in the membrane protein pattern are demonstrated. These alterations are caused by a mutation affecting a sugar transport system and by growth in the presence of D-fucose, inducer of the transport system. A further example of membrane protein changes introduced by growth at the nonpermissive temperature of a temperature-sensitive cell division mutant is shown. Finally, it is demonstrated that the major outer membrane component of Escherichia coli K-12 contains more than four proteins of similar molecular weight.     

Immunological detection of specific proteins in total cell extracts by fractionation in gels and transfer to diazophenylthioether paper

We describe a sensitive immunological procedure for the detection of specific proteins in total cell extracts and for the comparison of antigenically related polypeptides. Proteins are fractionated in polyacrylamide gels and transferred electrophoretically to diazophenylthioether paper, to which they bind covalently. Specific proteins are identified by incubation with specific antibody and 125 I-labeled protein A from Staphylococcus aureus, followed by autoradiography. High-resolution separation of proteins prior to transfer is achieved by polyacrylamide gradient gel electrophoresis in the presence of sodium dodecyl sulfate or by nonequilibrium pH gradient electrophoresis, followed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Further information can be obtained by limited enzymatic proteolysis of the proteins in the gel following polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate and analysis of the cleavage products by gel electrophoresis at right angles to the first gel. We show the application of this technique to the detection and comparison in extracts from infected cells of proteins related immunologically to the simian virus 40 capsid proteins VP1 and VP3.     

Electroblotting onto glass-fiber filter from an analytical isoelectrofocusing gel: a preparative method for isolating proteins for N-terminal microsequencing

A new method has been developed for the isolation of proteins for microsequencing. Proteins were separated by isoelectric focusing on polyacrylamide slab gels. Ampholytes in the gel were washed out with 3.5% (v/v) perchloric acid, and the proteins were electroblotted onto unmodified glass-fiber sheets. The immobilized proteins on the glass-fiber sheet were detected with Coomassie blue dye staining. The protein bands were then excised from the sheet and inserted into a gas phase sequenator for direct sequencing. They could also be extracted with sodium dodecyl sulfate buffer for molecular weight determination. Bovine serum albumin, beta-lactoglobulin A, and soybean trypsin inhibitor have been used as standard proteins for the test of this technique. Using this technique, we have determined the partial N-terminal sequence (26 residues) of an acidic (pI 5.6) glutathione S-transferase isolated from the chicken liver.     

An improved horizontal slab gel electrophoresis apparatus for DNA separation

An improved horizontal slab gel electrophoresis apparatus was developed for the separation of DNA restriction fragments. The apparatus was designed for both analytical and preparative runs. The use of agarose or polyacrylamide wicks rather than paper wicks simplifies the use of and increases the capabilities of horizontal slab gel electrophoresis.