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.     

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.     

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.     

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.     

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.     

Analysis of electroblotted proteins by mass spectrometry: protein identification after Western blotting

We describe a new approach for the identification and characterization by mass spectrometry of proteins that have been electroblotted onto nitrocellulose. Using this method (Blotting and Removal of Nitrocellulose (BARN)), proteins can be analyzed either as intact proteins for molecular weight determination or as peptides generated by on-membrane proteolysis. Acetone is used to dissolve the nitrocellulose and to precipitate the adsorbed proteins/peptides, thus removing the nitrocellulose which can interfere with MS analysis. This method offers improved protein coverage, especially for membrane proteins, such as uroplakins, because the extraction step after in-gel digestion is avoided. Moreover, removal of nitrocellulose from the sample solution allows sample analysis by both MALDI- and (LC) ESI-based mass spectrometers. Finally, we demonstrate the utility of BARN for the direct identification of soluble and membrane proteins after Western blotting, obtaining comparable or better results than with in-gel digestion.     

SDS-PAGE characterization of the proteins in equine seminal plasma

The aims of this project were to document the protein profile of equine seminal plasma and determine the variability between stallions in the relative composition of proteins in the ejaculate. A single ejaculate was obtained from 14 stallions of varying breed and age. The gel fraction was removed by an in-line filter. The semen was centrifuged and the supernatant seminal plasma aspirated without disturbing the sperm pellet. The seminal plasma was recentrifuged and stored in cryovials at -70 degrees C. Samples were thawed, recentrifuged, assayed for protein concentration (BCA protein assay), divided into aliquots, then stored at -70 degrees C. A standard protein concentration of 50 microg was loaded in each 10 microl sample. SDS-PAGE was performed using 15% polyacrylamide and a mixture of molecular weight standards. The electrophoresed gel was stained for proteins with Coomassie blue, air-dried, then scanned by a megapixel camera interfaced to a computer. Image analysis software calculated integrated optical density (IOD) values for each lane, and bands within a lane. Each band IOD was expressed as a percentage of the total lane IOD, thus reflecting the relative concentration of each protein within the ejaculate. A total of 14 bands were identified, ranging from a large 120 kDa protein down to a small 14 kDa protein. No sample contained all 14 protein bands. Seven protein bands (101 kDa, 32 kDa, 26 kDa, 22 kDa, 18 kDa, 16 kDa, 14 kDa) were present in all samples, however the relative concentrations of protein within those bands varied between stallions. We demonstrated that although there is a characteristic equine seminal plasma protein profile on SDS-PAGE gels, there is between stallion variability in the relative amounts of each protein.     

Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane.

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.     

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.     

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.     

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.     

Separation of highly fluorescent proteins by SDS-PAGE in Acroporidae corals

This study characterized the spectral properties of Acropora tenuis, A. nasuta, A. secale, and A. aspera, all of which showed strong colorful fluorescence under ultraviolet light-A (black light). The emission maxima of fluorescence from the intact corals were 517, 482, 484, and 514 nm in A. tenuis, A. nasuta, A. secale, and A. aspera, respectively. Using a soluble fraction of cell-free extract of the corals, we applied a method of sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to separate each fluorescent protein component contained in the corals. Green fluorescent bands were detected in all Acropora examined, although their apparent molecular mass and relative content were different. A. aspera had two orange bands in addition to the green one. The major excitation and emission peaks of the orange fluorescence bands were almost identical (476 and 478 nm), however, they were discernible by their spectral profiles and molecular masses. Some biochemical properties of the highly fluorescent proteins of Acropora are described and implications of the results are discussed.     

Suppression of nonspecific binding of avidin-biotin complex (ABC) to proteins electroblotted to nitrocellulose paper

Nitrocellulose blots of cell extracts reacted in sequence with biotinylated lectins and horseradish peroxidase-labeled avidin-biotin complex (ABC) often show considerable nonspecific staining of protein bands. Experiments were performed to determine which of the components of the ABC were responsible for this and whether or not the nature and ionic strength of the buffer used could alter this binding. Furthermore, as powdered non-fat milk has been proposed as a possible blocking agent for nonspecific binding of ABC, we sought to determine if it would adequately block that binding in our system. The initial experiments showed that nonspecific binding of ABC to proteins transferred to nitrocellulose membranes was due to the avidin component of the ABC; little, if any, binding was seen if biotin alone was incubated with these blots. The spurious binding was shown to be primarily due to the high affinity of avidin to proteins electroblotted to nitrocellulose, when incubated in low-salt buffers. Low-fat milk added to the buffer reduced overall nonspecific reactivity but produced additional artifacts in the form of bands that were not seen in other preparations. Nonspecific avidin binding to proteins transferred to nitrocellulose can therefore be effectively reduced by adding extra salt to buffers, whereas the addition of non-fat dry milk does not seem suitable for this purpose.     

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.     

Comparison of Staphylococcus spp. cellular and extracellular proteins by SDS-PAGE

In this study, a total of fifteen staphylococcal strains belonging to different species were characterized by whole-cell and extracellular protein profiles using sodium dodecylsulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The results are presented as dendrograms after quantitative analysis of the band patterns with a computer program. Visual inspection of protein bands and cluster analysis of protein patterns of 15 strains representing 10 Staphylococcus species showed that whole-cell and extracellular protein profiles differed in several protein bands in Staphylococcus aureus, S. epidermidis, S. simulans and other species of Staphylococcus; however, the differences were insufficient for reliable differentiation of Staphylococcus species by the SDS-PAGE method.     

腰带长体茧蜂Macrocentrus cingulum毒液和卵巢蛋白的电泳分析

寄生蜂毒液和卵巢蛋白在寄生过程中起着重要作用,蛋白成分和性质的研究日益深入.本文主要通过SDS-聚丙烯酰胺凝胶电泳(SDS-PAGE)试验,分析了腰带长体茧蜂毒液和卵巢蛋白的分子量组成.结果表明,腰带长体茧蜂毒液蛋白图谱显示约有7条蛋白带介于43～100 kDa之间,含量较高的三条带为97kDa、64kDa、45kDa;卵巢蛋白图谱显示约有12条蛋白带,位于30～200 kDa之间,含量较高的两条带为39kDa、43kDa.并对毒液和卵巢的生物学功能进行了探讨.     

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.