Preparation of GDP-D-mannose specifically labeled with tritium in the D-mannosyl moiety

A method, called “bidirectional transfer”, has been described for the transfer of DNA and RNA from agarose or polyacrylamide gels onto diazobenzyloxymethyl (DBM)-paper or nitrocellulose filters. The gels were sandwiched between either two nitrocellulose filters or two diazobenzyloxymethyl-papers. Next, the nucleic acids were allowed to diffuse out of the gels onto the filters. In this way, duplicate blots were obtained from a single gel. The bidirectional transfer of DNA or RNA from 0.5 to 1% agarose gels was complete and nearly quantitative after 1 h of transfer. DNA fragments from 5% polyacrylamide gels were efficiently blotted after 36 h onto nitrocellulose filters using bidirectional transfer. The fragments were transferred with good resolution and were shown to be efficient substrates for homologous [³²P]DNA probes.     

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.     

Direct tissue isoelectric focusing on ultrathin polyacrylamide gels. Applications in enzyme, lectin and immunohistochemistry

Summary Application of cryostal sections directly onto ultrathin polyacrylamide gels and subsequent isoelectric focusing allows elution of proteins, glycoproteins and peptides out of the sections into the gels. The eluted compounds reveal clearly delineated band patterns in the polyacrylamide gels. The advantage of this method is that enzyme histochemical reactions can be directly performed in the gel and in the electroeluted tissue sections. Therefore, this method is suitable for specifying, in more detail, histochemical enzyme reactions and for detecting multiple forms of enzymes even from a single tissue section. Furthermore, the transfer of proteins, glycoproteins and peptides from the gel onto nitrocellulose by a modified Western blot procedure offers the possibility of checking findings obtained by lectin histochemistry and immunohistochemistry.     

Avidin- or streptavidin-biotin as a highly sensitive method to stain total protein on membranes

A sensitive method for staining proteins after transfer from polyacrylamide gels to nitrocellulose paper is described. Transferred proteins are first derivatized by reaction of the nitrocellulose replica with sulfosuccinimidobiotin and are then reacted sequentially with streptavidin, rabbit anti-streptavidin, and horseradish peroxidase-conjugated goat anti-rabbit IgG antibody. Incubation with the enzyme substrate α-chloronaphthol, produces dark protein bands against a white background. The binding of streptavidin to the proteins is dependent on biotin derivatization as demonstrated by competition with biotinylated bovine serum albumin or 10 nM biotin. The procedure detects less than 5ng of transferred protein in a single band and is thus 5–10 times more sensitive than horseradish peroxidase-conjugated avidin alone. For bovine serum albumin, the method is comparable in sensitivity to silver staining of protein in polyacrylamide gels.     

Western blots using stained protein gels.

A general method is described for the electrophoretic transfer of proteins from stained gels to membranes and subsequent Western detection of specific proteins on the stained membranes. Proteins are separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, and the gels are stained using either of two different methods followed by electrophoretic transfer to nitrocellulose or Immobilon-P membranes. The transferred proteins remain stained during immunodetection, providing a set of background markers for protein location and size determination.     

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.     

Electrotransfer of basic proteins from nondenaturing polyacrylamide acid gels to nitrocellulose: detection of enzymatic and inhibitory activities and retention of protein antigenicity.

We have developed a method for electrotransfer of strongly basic proteins (lysozyme, pI 11; mucus proteinase inhibitor, pI greater than 10; bovine pancreas trypsin inhibitor; pI 10.5; human leukocyte elastase, pI greater than 9) from nondenaturing acid gels (pH 4.5) to nitrocellulose sheets. Buffers were those used in a discontinuous system for transfer from sodium dodecyl sulfate (SDS)-containing polyacrylamide gels with one modification in the cathode buffer which contained 0.1% SDS. This method was compared to electrotransfer performed in 0.7% acetic acid. The basic proteins studied, which were positively charged in the gel, formed with SDS negative complexes which migrated toward the anode and were efficiently transferred to the nitrocellulose. Moreover, their biological properties were preserved: inhibitory activity, enzyme activity, and antigenicity. This method is advantageous because it is simple, is sensitive, and can be applied to various biological fluids to detect inhibitors, enzymes, and other proteins which have a basic character, after electrophoretic separation under their native forms.     

Identification of protein bands in polyacrylamide gel by protein printing

Previously described methods for identification of proteins separated in cylindrical polyacrylamide gels have been found to be costly in time and antiserum and difficult to apply to small amounts of protein as are found in cerebrospinal fluid. We describe a method which involves printing of the proteins on the cut surface of the gel onto nitrocellulose paper. The protein bands of the imprint can then be identified using labelled antibodies. We have found this to be economical and quick, and it has permitted sensitive and reliable identification of proteins in unconcentrated cerebrospinal fluid and aqueous humour.     

Electrophoretic transfer of proteins from fixed and stained gels

A procedure by which sodium dodecyl sulfate gels can be fixed and stained with Coomassie blue and subsequently transferred to nitrocellulose for immunostaining is outlined. The procedure involves the complete removal of the stain followed by equilibration of the gel in sodium dodecyl sulfate running buffer. The efficiency of transfer is comparable to unfixed gels and the protein pattern of the transfer appears to be sharper, presumably due to less diffusion during the transfer process. The procedure does not affect the antigenicity of the proteins that have been examined by subsequent immunostaining. This method is particularly useful for situations in which sample size or concentration are limiting factors resulting in insufficient material for duplicate gels.     

Identification of Neuron-Specific Enolase and Nonneuronal Enolase in Human and Rat Brain on Two-Dimensional Polyacrylamide Gels

The location of the enzymes neuron-specific enolase and nonneuronal enolase on two-dimensional gels generated from tissue samples obtained from fresh human and rat cortex has been identified. This identification is based upon the following criteria: comigration on polyacrylamide gels with the appropriate purified protein and staining on nitrocellulose protein blots of human and rat cortex using antibodies specific for each protein. The results show that our preparation of neuron-specific enolase from rat and human brain is highly pure, as only one spot is obtained on two-dimensional gels. Further, the antiserum to neuron-specific enolase is highly specific, as it reacts only with neuron-specific enolase on nitrocellulose blots derived from two-dimensional gels of cortical tissue. The location of these proteins is of interest because it positively identifies two major brain proteins on two-dimensional polyacrylamide gels of fresh cortical tissue. This information will be useful in a variety of future studies aimed at both identifying specific proteins on two-dimensional gels and observing the effects of experimental manipulations on brain and other neuronal proteins.     

Fast and efficient method for detection and estimation of proteins

A quick, simple, inexpensive and sensitive method is described to stain and quantitate proteins on nitrocellulose papers. The proteins may be spotted or transferred from polyacrylamide gels by Western blotting. The procedure involves non-radioactive iodination of the polypeptides by chloramine T and potassium iodide followed by detection of bound iodine with starch. The method is more sensitive and much quicker than Coomassie brilliant blue staining and may be used for quantitation or detection of proteins in unknown samples. Another major advantage of this procedure is that ionic or nonionic detergents, although at higher concentrations causing the sample to disperse more broadly in the membranes, do not affect the staining procedure. Further, this method may be used for detection of proteins bound to papers that have high affinity for proteins such as the Zeta probe membranes.     

Vertical slab gel electrophoresis on a large number of samples: an apparatus with the capacity for central cooling

Following horizontal electroelution, or blotting, of proteins from polyacrylamide gels to immobilizing matrices, such as nitrocellulose or Zeta-bind paper, the transferred proteins can be derivatized in situ with pyridoxal 5'-phosphate and sodium borohydride. After a quenching step to eliminate nonspecific binding of antibody to the protein-binding matrix, the blot is incubated with a solution containing a mouse monoclonal antibody specific for the 5'-phosphopyridoxyl group. The transferred proteins can then be located on the blot with second antibody staining procedures employing either a peroxidase-linked goat anti-mouse F(ab')2 antibody or a peroxidase-linked avidin/biotin system. The solid-phase enzyme-linked immunosorbent assay method described in this report is a mild, general, and sensitive immunochemical method for the detection of proteins on protein-binding matrices.     

Development of buffers for fast semidry transfer of proteins

Western blot is an extensively used method for protein detection in cell biology. To optimize this procedure, here we examined a panel of buffers for their ability to efficiently transfer proteins from SDS–polyacrylamide gels onto nitrocellulose membranes in a short 12-min period, designated here as fast semidry transfer. Our results show for the first time that HEPES- and HEPPS/EPPS-based buffers represent the most efficient buffers for fast semidry transfer.     

The 43-K protein, v1, associated with acetylcholine receptor containing membrane fragments is an actin-binding protein.

Acetylcholine receptor enriched membrane fragments were obtained from the electric organs of Torpedo marmorata. The purified membrane fragments contained several proteins in addition to the acetylcholine receptor subunits. One of these was shown to be actin by means of immune blotting with a monoclonal antibody. Brief treatment of the membranes with pH 11.0 buffer removed actin and the other non-receptor proteins including the receptor-associated 43 000 mol. wt. polypeptide. This polypeptide was shown to bind actin after transferring the proteins from one- and two-dimensional polyacrylamide gels to nitrocellulose paper and incubating the nitrocellulose blots with actin. Specifically bound actin was demonstrated using the monoclonal antibodies to actin. No calcium or calmodulin dependency of binding was observed. The findings suggest that the 43 000 mol. wt. polypeptide is a link between the membrane-bound acetylcholine receptor and the cytoskeleton.     

Demonstration of a high density lipoprotein (HDL)-binding protein in Hep G2 cells using colloidal gold-HDL conjugates

Solubilized membrane proteins of Hep G2 cells were electrophoretically separated on polyacrylamide gels and electrotransferred onto nitrocellulose paper. Overlaying the nitrocellulose with human high density lipoproteins conjugated to colloidal gold revealed the presence of a single protein band with an apparent molecular mass of 80 kDa. Binding of the conjugates to this protein was specific for high density lipoproteins in as much as it was effectively displaced by an excess of unlabelled high density lipoproteins but not by a similar excess of unlabelled low density lipoproteins. Binding was not dependent on Ca²⁺ as 10 mM EDTA had no effect. The binding activity of the solubilized membranes was increased by incubating the cells with non-lipoprotein cholesterol. This was detected on electroblots and quantified with a new dot blot assay using the colloidal gold-high density lipoprotein conjugates.     

Reversible staining and peptide mapping of proteins transferred to nitrocellulose after separation by sodium dodecylsulfate-polyacrylamide gel electrophoresis

We describe a staining technique, using Ponceau S in very mild conditions, by which proteins can be visualized on nitrocellulose replicas without being permanently fixed to the membrane itself, thus allowing subsequent procedures such as immunoblotting or preparative elution of the proteins to be performed. This staining technique can detect 250 to 500 ng protein, which is essentially the same sensitivity seen for Coomassie blue staining of proteins on nitrocellulose. The Ponceau S staining technique was used to locate proteins on nitrocellulose replicas for subsequent in situ radioiodination and trypsin digestion, followed by separation of the resultant digests in two-dimensional peptide analysis. Staining proteins with Ponceau S did not interfere with either the radioiodination or trypsin digestion, as indicated by essentially identical peptide patterns being obtained for the internal protein p26 from equine infectious anemia virus, regardless of whether the digests were prepared from polyacrylamide gel slices or nitrocellulose sections. The combination of preparation of radioiodinated tryptic digests on nitrocellulose and subsequent two-dimensional analysis is sensitive enough to detect peptide additions and deletions occurring in the surface antigen gp90 recovered from two antigenically distinct strains of equine infectious anemia virus. Thus these procedures provide a relatively simple, inexpensive, and highly reproducible technique for the analysis of as little as 250 nanograms of protein after separation by electrophoresis in polyacrylamide gels.     

High mobility group proteins 1 and 2 are present in simian virus 40 provirions, but not in virions.

Viral particles at the late stages of SV40 morphogenesis were examined for the presence of HMG proteins 1 and 2, by an immunochemical method involving the transfer of proteins from polyacrylamide gels to nitrocellulose membranes. It was found that these proteins are present in SV40 provirions, in which histone H1 is still associated with viral chromatin, but absent in mature SV40 virions.     

Direct tissue isoelectric focusing on mini ultrathin polyacrylamide gels followed by subsequent western blotting, enzyme detection, and lectin labeling as a tool for enzyme characterization in histochemistry.

Application of cryostat sections directly onto mini ultrathin polyacrylamide isoelectric focusing gels allows an elution of proteins out of the sections into the gels under conventional focusing conditions. Protein bands representing alkaline phosphatases can easily be identified on nitrocellulose after performing a modified Western blot procedure. Furthermore, carbohydrate residues of several isoforms of alkaline phosphatases separated by isoelectric focusing can be demonstrated in a single blot strip by subsequent incubation of this strip with substrates for alkaline phosphatase and peroxidase, the latter being employed as the enzyme to which the applied lectins are covalently linked. This simple and reproducible procedure is likely to enable histochemists to determine isoforms of enzymes from a single cryostat section.     

Electrotransfer of proteins in an environmentally friendly methanol-free transfer buffer

Western blot transfer buffer was modified to substitute the acute poison methanol, with the common rubbing alcohol, isopropanol in concentrations of as low as 5 % for protein electrotransfer. Commercially available molecular weight markers and rabbit serum were run on polyacrylamide gels and shown to be transferred adequately to both nitrocellulose and polyvinylidene difluoride membranes under either wet or semi-dry conditions with similar results in all cases. This procedure was successfully used for immunodetection of the rabbit IgG heavy chain from serum. Therefore, this represents a good alternative for less toxic and environmentally friendly conditions for western immunoblotting of proteins.     

Double replica electroblotting: a method to produce two replicas from one gel

Electroblotting is a method by which proteins or nucleic acids, separated by electrophoresis, are transferred, also by electrophoresis, from a gel to a so-called transfer medium, e.g a nitrocellulose membrane. In some experiments, it is desirable to be able to obtain more than one replica from each gel and it has now proved possible to produce two replicas, which are almost identical, from one gel. This is achieved by applying one membrane on each side of the gel and change the direction of the current several times in such a way that the efficient transfer time is short in the beginning of the electroblotting and is increased for each cycle. This procedure will be referred to as 'double replica electroblotting'. Proteins were transferred at 100 V and the duration of an experiment with 2 h efficient transfer time in each direction was 7 h. The gel was more efficiently depleted of proteins after double replica electroblotting as compared to ordinary electrotransfer in one direction. Cathodically migrating proteins are also trapped on the membranes with this technique. Double replica electroblotting was used to produce two replicas from ordinary sodium dodecyl sulfate polyacrylamide gels as well as from 2-dimensional gels.