Direct amino acid analysis of proteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel

The band of appropriate proteins (basic pancreatic trypsin inhibitor, soybean trypsin inhibitor, interleukin 2, and human leukocyte interferon alpha A) on a polyvinylidene difluoride (PVDF) membrane, which was electroblotted from sodium dodecyl sulfate (SDS)-polyacrylamide gel and then stained with Coomassie blue R-250, was cut out and directly hydrolyzed in HCl in the presence of thioglycolic acid for amino acid analysis. The analytical values agreed with those expected with recoveries of 29-47%, except that the value for tryptophan was very low or scarcely detected. This method was applied to the identification of human growth hormone (hGH) in a partially purified preparation. The amino acid composition of the band corresponding to about 2 micrograms of hGH agreed with the theoretical values. These results indicate that the band on the PVDF membrane can be directly hydrolyzed for amino acid analysis and that the method can be used for partially purified proteins separated using SDS-polyacrylamide gel electrophoresis.     

Imidazole-SDS-Zn reverse staining of proteins in gels containing or not SDS and microsequence of individual unmodified electroblotted proteins.

A reverse staining procedure is described for the detection of proteins in acrylamide and agarose gels with and without SDS. Protein detection occurs a few minutes after electrophoresis. The sensitivity on acrylamide gels is higher than that of Coomassie blue staining either on acrylamide gels or on electrotransferred membranes. Sequencing of protein bands only detected by reverse staining on the gel and not by Coomassie blue is demonstrated.     

Microsequence analysis of electroblotted proteins. I. Comparison of electroblotting recoveries using different types of PVDF membranes.

The most effective protein purification method of low picomole amounts for sequence analysis involves polyacrylamide gel electrophoresis followed by electroblotting to polyvinylidene difluoride (PVDF) membranes. Since a critical factor in this procedure is the protein recovery at the blotting step, different types of PVDF membranes were systematically evaluated for their ability to bind proteins during electrotransfer. Differences in electroblotting recoveries occurred between types of PVDF membranes for some proteins. Some variability persisted even when optimized electroblotting procedures were used which reduce the sodium dodecyl sulfate (SDS) concentration in the gel and improve protein-PVDF binding. The membranes which were evaluated could be grouped as either "high retention" membranes (ProBlott, Trans-Blot, and Immobilon-PSQ) or "low retention" membranes (Immobilon-P and Westran). The high retention membranes showed higher protein recoveries under most conditions tested, especially for small proteins or peptides. These high retention membranes were also less sensitive to the exact electroblotting conditions, especially those factors which affect the amount of SDS present during either electrotransfer or direct adsorption from protein solutions. High retention PVDF membranes are therefore preferred in most cases for optimal protein or peptide recovery prior to direct sequence analysis. In contrast, low retention membranes are preferred for procedures where subsequent extraction of the proteins from the membranes is required. Even under identical conditions, substantial protein-to-protein variation for both adsorption and subsequent extraction is routinely observed for both groups of membranes, indicating that the nature of protein-PVDF interactions is more complex than simple hydrophobic interactions.     

Solid-phase sequence analysis of proteins electroblotted or spotted onto polyvinylidene difluoride membranes

Electroblotted proteins noncovalently bound to polyvinylidene difluoride (PVDF) membranes are typically sequenced using adsorptive sequencer protocols (gas-phase or pulsed-liquid) that do not require a covalent linkage between protein and surface. We have developed simple chemical protocols where proteins are first electroblotted onto unmodified PVDF membranes, visualized with common protein stains, and then immobilized for solid-phase sequence analysis. Adsorbed, stained proteins are first treated with phenylisothiocyanate (PITC) to modify alpha and epsilon amines. The protein is then overlayed with a solution of 1,4-phenylene di-isothiocyanate (DITC), followed by a few microliters of a basic solution containing a poly(alkylamine). As the polymer dries onto the surface both polymer and remaining protein amino groups are crosslinked by DITC. The protein is thus immobilized to the membrane surface by entrapment in a thin polymer coating. The coating is transparent to the degradation chemistry, and extensive enough to remain immobilized even in the absence of any covalent link between polymer and surface. Partial modification with PITC allows for identification of N-terminal and internal lysine residues during sequencing. The process was tested with a variety of poly(alkylamines), linear and branched, with molecular weights ranging from 600 to over 100,000. Proteins bound in this manner were successfully sequenced using covalent (solid-phase) sequencer protocols with cycle times as short as 26 min.     

Microsequence analysis of electroblotted proteins. II. Comparison of sequence performance on different types of PVDF membranes.

The influence of different types of polyvinylidene difluoride (PVDF) membranes on gas phase sequence performance has been evaluated. These PVDF membranes have been classified as either high retention (Trans-Blot and ProBlott) or low retention membranes (Immobilon-P) based on their ability to bind proteins during electroblotting from gels. Initial yields, repetitive yields, and extraction efficiency of the anilinothiazolinone amino acid derivatives have been compared for several standard proteins that have been either electroblotted or loaded onto PVDF membranes by direct adsorption. These results show that the major differences in initial sequence yields between membranes arise from differences in the amount of protein actually transferred to the membrane rather than sequencer-related factors. In contrast to several previous observations from other laboratories, more tightly bound proteins do not sequence with lower initial yields and initial yields are not affected by the ratio of surface area to protein. The stronger binding on high retention PVDF membranes does not adversely affect recoveries of difficult to extract, or very hydrophobic, amino acid derivatives. Several amino acids, especially tryptophan, are actually recovered in dramatically higher yield on high retention membranes compared with either Immobilon or glass filters. At the same time, the protein and peptide binding properties of high retention membranes will frequently improve the repetitive yield by minimizing sample extraction during the sequencer cycle. Stronger protein binding together with improved electroblotting yields offer substantially improved sequence performance when high retention PVDF membranes are used.     

Direct carbohydrate analysis of glycoproteins electroblotted onto polyvinylidene difluoride membrane from sodium dodecyl sulfate-polyacrylamide gel.

A procedure for the carbohydrate analysis of glycoproteins electrotransferred to a polyvinylidene difluoride membrane is described. The glycoproteins (plant lectins, transferrin, and vitronectin) were first separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and then electroblotted onto a membrane. Each of the glycoprotein bands visualized by staining with Coomassie brilliant blue R-250 was excised from the membrane and subjected to direct hydrolysis either in 2.5 M trifluoroacetic acid at 100 degrees C for 6 h for neutral sugars and hexosamines, or in 0.05 M H2SO4 at 80 degrees C for 1 h for sialic acids. The hydrolysate obtained was analyzed for neutral sugars, hexosamines, and sialic acids independently by three different systems of high-performance liquid chromatography. The analytical values were reproducible with reasonable accuracy and agreed with those expected with recoveries of 57-66%. The method was successfully applied to a mannose-specific lectin of Sophora japonica bark, which is composed of four different subunits that aggregate sugar specifically. Because the four subunits could be separated by SDS-PAGE alone, the method proved useful for determining their carbohydrate compositions. Three of them were shown to contain carbohydrates typical of N-linked oligosaccharides of plant origin, which agreed well with the results of the binding assay carried out on a membrane using various horseradish peroxidase-labeled lectins.     

Sequence from picomole quantities of proteins electroblotted onto polyvinylidene difluoride membranes

Small amounts (7-250 pmol) of myoglobin, beta-lactoglobulin, and other proteins and peptides can be spotted or electroblotted onto polyvinylidene difluoride (PVDF) membranes, stained with Coomassie Blue, and sequenced directly. The membranes are not chemically activated or pretreated with Polybrene before usage. The average repetitive yields and initial coupling of proteins spotted or blotted into PVDF membranes ranged between 84-98% and 30-108% respectively, and were comparable with the yields measured for proteins spotted onto Polybrene-coated glass fiber discs. The results suggest that PVDF membranes are superior supports for sequence analysis of picomole quantities of proteins purified by gel electrophoresis.     

Micro-determination of amino acid composition of proteins electroblotted onto polyvinylidene difluoride membranes

A method for determination of amino acid composition of proteins separated by SDS-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride (PVDF) membranes is described. A single blotted band containing 50 to 200 pmoles of protein was cut out and submitted to acid hydrolysis with HCl followed by derivatization with phenylisothiocyanate. The amino acid derivatives were separated by reverse phase high-performance liquid chromatography. Bovine serum albumin, lysozyme, myoglobin, ovalbumin, soybean trypsin inhibitor and carbonic anhydrase were analyzed; the results revealed a good correspondence with reported values. This can be considered an analytical method to determine the amino acid composition of samples from microquantities of protein mixtures, particularly in those cases in which SDS-polyacrylamide gel electrophoresis is the most suitable separation system.     

Functionalized membrane supports for covalent protein microsequence analysis

Methods were developed for high yield covalent attachment of peptides and proteins to isothiocyanate and arylamine-derivatized poly(vinylidene difluoride) membranes for solid-phase sequence analysis. Solutions of protein or peptide were dried onto 8-mm membrane disks such that the functional groups on the surface and the polypeptide were brought into close proximity. In the case of the isothiocyanate membrane, reaction between polypeptide amino groups and the surface isothiocyanate moieties was promoted by application of aqueous N-methylmorpholine. Attachment of proteins and peptides to the arylamine surface was achieved by application of water-soluble carbodiimide in a pH 5.0 buffer. Edman degradation of covalently bound polypeptides was accomplished with initial and repetitive sequence yields ranging from 33 to 75% and 88.5 to 98.5%, respectively. The yields were independent of the sample load (20 pmol to greater than 1 nmol) for either surface. Significant loss of material was not observed when attachment residues were encountered during sequence runs. Application of bovine beta-lactoglobulin A chain, staphylococcus protein A, or the peptide melittin to the isothiocyanate membrane allowed for extended N-terminal sequence identification (35 residues from 20 pmol of beta-lactoglobulin). A number of synthetic and naturally occurring peptides were sequenced to the C-terminal residue following attachment to the arylamine surface. In one example, 10 micrograms of bovine alpha-casein was digested with staphylococcal protease V8 and the peptides were separated by reverse-phase chromatography. Peptide fractions were then directly applied to arylamine membrane disks for covalent sequence analysis. From as little as 2 pmol of initial signal it was possible to determine substantial sequence information (greater than 10 residues).     

High-yield recovery of electroblotted proteins and cleavage fragments from a cationic polyvinylidene fluoride-based membrane.

In this report we describe the use of a novel, experimental, polyvinylidene fluoride-based membrane with a cationic surface for the isolation by electroblotting of small amounts of proteins separated by gel electrophoresis for further characterization by protein fragmentation for internal sequence analysis. The membrane is characterized by a surface that mediates primarily ionic protein/membrane interactions and that allows the recovery of adsorbed proteins at high yields under relatively mild conditions. In electroblotting experiments, the novel membrane has a binding capacity that is at least equivalent to that of standard polyvinylidene fluoride membranes and is compatible with both chemical and enzymatic fragmentation of blotted proteins in situ. Intact electroblotted proteins, or fragments thereof, were eluted at high yields. Further structural analysis is demonstrated using reverse-phase high-performance liquid chromatography or gel electrophoresis to separate cleavage fragments for either pulsed-liquid- or solid-phase automated sequence analysis.     

Manual precyclization of fiberglass filters for microsequence analysis of peptides and proteins

A manual procedure for precyclization of fiberglass filters has been described. The quality of the manually precycled filter is as good for microsequence analysis of proteins and peptides as a filter that has been precycled by an automatic system. The manually precycled filter is convenient to use and saves time and money.     

A new method of detecting hormone-binding proteins electroblotted onto glass fiber filter: juvenile hormone-binding proteins from grasshopper hemolymph

We have developed a new method to identify juvenile hormone (JH)-binding proteins blotted onto glass fiber filter (GFF) after electrophoretic separation. Insect JH regulates reproduction in the two-striped grasshopper, Melanoplus bivittatus. A number of proteins are involved in the delivery of JH from its site of synthesis to the nuclei of fat body cells where it acts to induce vitellogenesis. To identify JH binding proteins, hemolymph was separated by PAGE, electroblotted onto GFF, and incubated in [10-3H]JH-III. The amount of hormone bound by blotted proteins increased with the amount of protein on the filter, was competitively displaced by excess non-labeled hormone, and was affiliated with individual bands on fluorograms of proteins blotted after electrophoretic separation. GFF etched with trifluoroacetic acid was better than nitrocellulose, Zeta Probe, cellulose acetate or unetched GFF. Phosphate (pH 6.0-7.3) or Tris buffers (pH 7.3-8.0) worked equally well for the procedure. Unbound hormone was easily removed by short washes in buffer, and adequate binding for detection was achieved in a 15 min incubation. Preliminary data suggest that this technique may be used to detect receptors, carriers, and binding proteins of steroid hormones.     

Microsequence analysis of winged bean seed proteins electroblotted from two-dimensional gel

Electroblotting method employing a semidry blotting apparatus for the subsequent protein microsequence analysis (Hirano, 1987) was improved. This method is convenient and allows rapid and efficient transfer of the proteins from a polyacrylamide gel (1 mm thick) onto the Polybrene-coated glass-fiber sheet or polyvinylidene difluoride membrane filter in only 20 min. The electroblotted proteins could be sequenced directly with the gas-phase protein sequencer at a 20-pmole level. This method was applied to the sequence analysis of winged bean seed proteins. A portion of the crude extracts from only one-twentieth of a seed of the winged bean was separated by two-dimensional polyacrylamide gel electrophoresis and electroblotted, and the N-terminal amino acid sequences of the blotted proteins were analyzed. The sequences of about 60% of the blotted major proteins, including nine Kunitz trypsin inhibitor-like proteins with heterogeneity in the N-terminal sequences, a protein that has a homologous sequence to the leghaemoglobin, nitrogen-fixing root nodule-specific protein, and a soybean basic 7S globulin-like protein could be easily identified.     

A new siliconized-glass fiber as support for protein-chemical analysis of electroblotted proteins

A new hydrophobic glass-fiber support is presented, which is well suited to the electrophoretic transfer of proteins from polyacrylamide gels and subsequent protein-chemical analysis. Modified glass-fiber sheets are easily prepared by chemical reaction of the surface with poly(methyl-3,3,3-trifluoropropylsiloxane) in trifluoroacetic acid. The modification is stable during electroblotting, amino acid sequence analysis and hydrolysis. The siliconized glass fiber exhibits a high protein-binding capacity, allows the application of well-established staining procedures, and does not interfere with the analytical methods of modern protein chemistry at the low picomole level. Samples separated by electrophoresis and immobilized on hydrophobic supports fail to exhibit any detectable contamination in amino acid sequence analysis hence allowing the high performance of the available protein-chemical methods to be exploited.     

Automated microsequence analysis by use of radioactive phenylisothiocyanate.

The use of radioactive phenylisothiocyanate as a coupling reagent in conjunction with an automated protein sequenator permits N-terminal sequence analysis of 1.5 nmoles of protein. This procedure should facilitate chemical studies on several membrane proteins of current interest (e.g., histocompatability and Ir-associated alloantigens) which are available in only small quantities.     

Purification of membrane proteins in SDS and subsequent renaturation

A prerequisite for the purification of any protein to homogeneity is that the protein is not non-specifically associated with other proteins especially during the final stage(s) of the fractionation procedure. This requirement is not so often fulfilled when nonionic detergents (for instance Triton X-100) are used for solubilization of membrane proteins. The reason is that these detergents are not efficient enough to prevent the protein of interest from forming aggregates with other proteins upon contact with chromatographic or electrophoretic supporting media, which, due to their polymeric nature, have a tendency to induce aggregation of other polymers, for instance, hydrophobic proteins. The aggregation can be avoided if sodium dodecyl sulfate (SDS) is employed as detergent. We therefore suggest that membrane proteins should be purified by conventional methods in the presence of SDS and that the purified proteins, which are in a denatured state, are allowed to renature. There is good change to renature internal membrane proteins since they should not be so susceptible to denaturation by detergents as are water-soluble proteins because the natural milieu of the former proteins is lipids which in fact are detergents. In this paper we present a renaturation method based on the removal of SDS by addition of a large excess of G 3707, a nonionic detergent. By this technique we have renatured a 5'-nucleotidase from Acholeplasma laidlawii and a neuraminidase from influenza virus. The enzyme activities were higher (up to 6-fold) after the removal of SDS than prior to the addition of SDS.