Detection of cellulose-binding proteins in electrophoresis gels by filter paper affinity blotting

Proteins separated in electrophoresis gels were tested for the ability to bind cellulose by a simple blotting procedure. Proteins were blotted onto Whatman No. 1 filter paper by diffusion or by electrophoretic transfer and detected by Coomassie blue staining. Certain proteins released into culture supernatant by Bacteroides succinogenes NR9 (ATCC 43854) adhered strongly to cellulose, but were not found to have carboxymethylcellulose activity. Boiling of samples prior to electrophoresis eliminated the ability of proteins to bind to cellulose. Proteins that did not adhere to filter paper cellulose were detected on a nitrocellulose membrane placed behind the filter paper during electrophoretic transfer. The technique, referred to as filter paper affinity blotting, detects cellulose-binding proteins with great sensitivity.     

A specific stain for the detection of nonheme iron proteins in polyacrylamide gels.

Nonheme iron proteins can be visualized as blue bands in native polyacrylamide gels using a staining method that is both simple and rapid. The reaction of potassium ferricyanide with protein-bound iron atoms to form royal blue complexes occurs almost instantaneously and is sensitive enough to detect 1 microgram of analytical-grade ferritin and 2 micrograms of purified ferredoxin from cyanobacteria. No special treatment of reagents or apparatus was necessary. On comparison, this stain was found to be more specific than the Ferene S stain, not detecting bovine serum albumin even when present as a hundredfold excess over ferritin. The method was found to be effective for isoelectric focusing gels as well.     

Immunological detection of N-formylkynurenine in oxidized proteins

Reactions of tryptophan residues in proteins with radical and other oxidative species frequently lead to cleavage of the indole ring, modifying tryptophan residues into N-formylkynurenine (NFK) and kynurenine. Tryptophan modification has been detected in physiologically important proteins and has been associated with a number of human disease conditions. Modified residues have been identified through various combinations of proteomic analyses, tryptic digestion, HPLC, and mass spectrometry. Here we present a novel, immunological approach using polyclonal antiserum for detection of NFK. The specificity of our antiserum is confirmed using photooxidation and radical-mediated oxidation of proteins with and without tryptophan residues. The sensitivity of our antiserum is validated through detection of NFK in photooxidized myoglobin (two tryptophan residues) and in carbonate radical-oxidized human SOD1, which contains a single tryptophan residue. Analysis of photooxidized milk also shows that our antiserum can detect NFK residues in a mixture of proteins. Results from mass spectrometric analysis of photooxidized myoglobin samples corroborate the immunological data, detecting an increase in NFK content as the extent of photooxidation increases.     

Ultrasensitive fluorescence-based detection of nascent proteins in gels

The most common method of analysis of proteins synthesized in a cell-free translation system (e.g., nascent proteins) involves the use of radioactive amino acids such as [(35)S]methionine or [(14)C]leucine. We report a sensitive, nonisotopic, fluorescence-based method for the detection of nascent proteins directly in polyacrylamide gels. A fluorescent reporter group is incorporated at the N-terminus of nascent proteins using an Escherichia coli initiator tRNA(fmet) misaminoacylated with methionine modified at the alpha-amino group. In addition to the normal formyl group, we find that the protein translational machinery accepts BODIPY-FL, a relatively small fluorophore with a high fluorescent quantum yield, as an N-terminal modification. Under the optimal conditions, fluorescent bands from nanogram levels of in vitro-produced proteins could be detected directly in gels using a conventional UV-transilluminator. Higher sensitivity ( approximately 100-fold) could be obtained using a laser-based fluorescent gel scanner. The major advantages of this approach include elimination of radioactivity and the rapid detection of the protein bands immediately after electrophoresis without any downstream processing. The ability to rapidly synthesize nascent proteins containing an N-terminal tag facilitates many biotechnological applications including functional analysis of gene products, drug discovery, and mutation screening.     

Citric acid extracts a specific set of proteins from isolated cell nuclei

The treatment of isolated cell nuclei with citric acid was described as a method for separating inner and outer nuclear membrane. Using cell nuclei from bovine cerebral cortex, we can show that citric acid does not cause a separation of the two nuclear membranes, but extracts a specific set of proteins from the nuclei. The extraction of proteins is not just an effect of damaging the nuclear membrane or destructing the cytoskeleton, but rather a specific effect of citric acid treatment. One of the extracted proteins, chosen as a marker for the putative outer nuclear membrane fraction, has an apparent molecular weight of 145 kDa and is located in the nucleoplasm as shown by immunofluorescence microscopy. By sequencing tryptic peptides it was identified as RNA helicase A, an abundant nuclear protein assumed to participate in the processing of mRNA. © 1995 Wiley-Liss, Inc.     

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.     

Quantitation of specific proteins in polyacrylamide gels by the elution of fast green FCF

The quantitation of proteins in polyacrylamide gels stained with Fast Green FCF has been investigated using a modification of the elution technique originally described by Fenner et al. (Fenner, C., Traut, R.R., Mason, D.T. and Wikman-Coffelt, J. (1975) Anal. Biochem. 63, 595–602) for Coomassie Blue and adapted by Medugorac (Medugorac, I. (1979) Basic Res. Cardiol. 74, 406–416) for use with proteins stained with Fast Green FCF. The elution of dye from stained protein was accomplished using 1.0 M NaOH instead of aquoeus pyridine as required by the original method. The primary advantages of our modification are that the time required for protein quantitation has been considerably reduced and the use of toxic organic solvents has been eliminated. We have investigated the applicability of the method to several different proteins and our results indicate: (a) The quantity of Fast Green eluted from specific proteins is proportional to the quantity of protein applied to the gel, but varies for each individual protein. (b) The method allows quantitation over a very wide range of protein (1–800 μg). (c) Quantitation of protein is independent of the width of the stained bands as well as acrylamide concentration. (d) The method is applicable to gels of many types including disc, slab and continuous gradient gel, (e) Protein can be estimated from the patterns obtained by two-dimensional polyacrylamide gel electrophoresis. (f) The presence of Triton X-100 in gel and protein sample does not affect quantitation; the method is applicable to gels containing SDS provided that SDS is removed prior to staining. (g) Precipitation of protein with 12.5% TCA following electrophoresis does not interfere with quantitation. (h) The reproducibility of the technique is excellent, with standard deviations being less than 10% of the mean in all cases. This method appears highly versatile but requires appropriate standards for the quantitation of individual proteins.     

Ultrasensitive silver-stain method for the detection of proteins in polyacrylamide gels and immunoprecipitates on agarose gels

The highly sensitive silver-stain procedure for the detection of proteins in polyacrylamide gels has been revised and simplified using a single-step silver ion reduction after suitable treatment of proteins with bifunctional aldehyde. Washing steps were eliminated and excellent reproducibility of results was achieved. Sensitivity obtained using this procedure was at least equal to that obtained with the original one. Use of the present silver-staining methods has been extended to the quantitative analysis of immunoprecipitates on agarose gels, with a good increase of sensitivity and excellent increase of resolution when compared to the Coomassie blue stain.     

Visible fluorescent detection of proteins in polyacrylamide gels without staining

2,2,2-Trichloroethanol (TCE) incorporated into polyacrylamide gels before polymerization provides fluorescent visible detection of proteins in less than 5min of total processing time. The tryptophans in proteins undergo an ultraviolet light-induced reaction with trihalocompounds to produce fluorescence in the visible range so that the protein bands can be visualized on a 300-nm transilluminator. In a previous study trichloroacetic acid or chloroform was used to stain polyacrylamide gel electrophoresis (PAGE) gels for protein visualization. This study shows that placing TCE in the gel before electrophoresis can eliminate the staining step. The gel is removed from the electrophoresis apparatus and placed on a transilluminator and then the protein bands develop their fluorescence in less than 5min. In addition to being rapid this visualization method provides detection of 0.2microg of typical globular proteins, which for some proteins is slightly more sensitive than the standard Coomassie brilliant blue (CBB) method. Integral membrane proteins, which do not stain well with CBB, are visualized well with the TCE in-gel method. After TCE in-gel visualization the same gel can then be CBB stained, allowing for complementary detection of proteins. In addition, visualization with TCE in the gel is compatible with two-dimensional PAGE, native PAGE, Western blotting, and autoradiography.     

The chemical fractionation of soil zinc and its specific and total adsorption by Egyptian alluvial soils

Summary The Zn contents of twenty-nine alluvial soils from Egypt were chemically fractionated into: water soluble+exchangeable, weakly bound to inorganic sites, organically bound, occluded as free oxide material, and residual mainly in the mineral structure. On the average these fractions constituted about 0.01, 1.20, 28.6, 21.5 and 45.5% of the total soil Zn respectively which averaged 76.25 ppm. Significant correlations were obtained between each individual Zn-fraction and some soil variable.Zinc adsorption isotherms were developed for seven soils suspended in dilute ZnCl₂ solution in the presence of either 0.05M CaCl₂ solution (Specific adsorption) or deionized water (Total adsorption). The Langmuir constants (adsorption maximum and bonding energy) were calculated. The average value of specific adsorption maximum was 1.94 mg Zn/g soil and of total adsorption maxima was 11.54 mg Zn/g soil. Correlation analysis showed that CEC, free Fe₂O₃ and clay content were the dominant soil variables contributing towards specific Zn adsorption. The (Zn) (OH)₂ ion concentration products in the solutions when Zn adsorption corresponded to the Langmuir adsorption maxima were 0.92×10^–17 in the specific adsorption treatment, and 1.35×10^–15 in the total adsorption treatment. These values are within the solubility range of Zn (OH)₂ and ZnCO₃. The values of Langmuir bonding energy constants showed that Zn was more strongly adsorbed by low carbonate or carbonate-free soils than by carbonate-rich soils.     

A specific iron stain for iron-binding proteins in polyacrylamide gels: application to transferrin and lactoferrin

A new method for specifically staining the iron atoms present in transferrin and lactoferrin after polyacrylamide gel electrophoresis and isoelectric focusing is described. The stain, 3-(2-pyridyl)-5,6-bis(2-(5-furylsulfonic acid))-1,2,4-triazine, disodium salt, or Ferene S, will detect transferrin in 5 microliters of human serum, lactoferrin in 10 microliters of human whey, and 10 micrograms of purified primate (Macaca fascicularis) transferrin. This method of staining is very rapid as the serum transferrin bands can be seen within 5 to 10 min of staining.     

Immunological characterization of microtubule-associated proteins specific for the immature brain

Immunoblotting analysis was used to detect the microtubule-associated proteins present at different stages of rat brain development. Polyclonal antibodies were raised against the two main adult brain microtubule-associated proteins: MAP-2 (300 kDa) and TAU (60-70 kDa). Whatever the stage of development, anti-MAP-2 serum detected high molecular mass proteins and at immature stages a protein of 62 kDa. This protein which has previously been referred to as 'young TAU slow' is, therefore, immunologically related to MAP-2. The anti-TAU serum (but not the anti-MAP-2 serum) detected at immature stages of development a 48 kDa protein which also disappears at adulthood. This 48 kDa entity which has been referred to as 'young TAU fast' is progressively replaced by the closely spaced bands (60-70 kDa) of adult TAU proteins. The 62 and 48 kDa proteins appear therefore to be immunologically distinct and represent two microtubule-associated proteins specific to the immature brain.     

Binding of proteins to specific target sites in membranes measured by total internal reflection fluorescence microscopy

A new quantitative technique for measuring the binding of proteins to membranes is described. The method is based on a combination of total internal reflection fluorescence microscopy and the preparation of supported planar bilayers. Specific and reversible binding of a fluorescence-labeled monoclonal antibody to lipid haptens that were embedded in supported bilayers has been measured by this technique and compared to binding experiments that were conducted on membrane vesicles in solution. Equilibrium binding constants and kinetic parameters have been determined and used to expand the picture of the antibody-lipid hapten reaction. Estimates demonstrate that this technique is capable of measuring a broad range of binding constants (down to about 10(4) M-1) using only small amounts of ligand and receptor.     

Rapid detection and quantification of specific proteins by immunodepletion and microfluidic separation

Conventional immunoblotting techniques are labor intensive, time consuming and rely on the elution of target protein after depletion. Here we describe a new method for detection and quantification of proteins, independent of washing and elution. In this method, the target protein is first captured by immunodepletion with antibody-coated microbeads. In the second step, both the supernatant after immunodepletion and the untreated protein sample are directly analyzed by microfluidic electrophoresis without further processing. Subsequently, the detection and quantification are performed by comparing the electropherograms of these two samples. This method was tested using an Escherichia coli lysate with a FLAG-tagged protein and anti-FLAG magnetic beads. An incubation of as short as one min was sufficient for detectable depletion (66%) by microchip electrophoresis. Longer incubation (up to 60 min) resulted in more depletion of the target band (82%). Our results show that only 19% of the target is recovered after elution from the beads. By eliminating multiple wash and elution steps, our method is faster, less labor intensive, and highly reproducible. The target protein can still be easily identified even in the case of nonspecific binding at low concentrations. This work highlights the advantages of integrating immunodepletion techniques on a microfluidic platform.     

T cell responses to known allergen proteins are differently polarized and account for a variable fraction of total response to allergen extracts

A panel of 133 allergens derived from 28 different sources, including fungi, trees, grasses, weeds, and indoor allergens, was surveyed utilizing prediction of HLA class II-binding peptides and ELISPOT assays with PBMC from allergic donors, resulting in the identification of 257 T cell epitopes. More than 90% of the epitopes were novel, and for 14 allergen sources were the first ever identified to our knowledge. The epitopes identified in the different allergen sources summed up to a variable fraction of the total extract response. In cases of allergens in which the identified T cell epitopes accounted for a minor fraction of the extract response, fewer known protein sequences were available, suggesting that for low epitope coverage allergen sources, additional allergen proteins remain to be identified. IL-5 and IFN-γ responses were measured as prototype Th2 and Th1 responses, respectively. Whereas in some cases (e.g., orchard grass, Alternaria, cypress, and Russian thistle) IL-5 production greatly exceeded IFN-γ, in others (e.g., Aspergillus, Penicillum, and alder) the production of IFN-γ exceeded IL-5. Thus, different allergen sources are associated with variable polarization of the responding T cells. The present study represents the most comprehensive survey to date of human allergen-derived T cell epitopes. These epitopes might be used to characterize T cell phenotype/T cell plasticity as a function of seasonality, or as a result of specific immunotherapy treatment or varying disease severity (asthma or rhinitis).     

Electrophoretic transfer of proteins and nucleic acids from slab gels to diazobenzylozymethyl cellulose or nitrocellulose sheets

A new electrophoretic technique for transferring proteins and nucleic acids from either polyacrylamide or agarose slab gels has been developed as an alternative to blotting. The method is fast, efficient and versatile. Efficient transfer of DNA to both nitrocellulose membrane and diazobenzyloxymethyl cellulose as well as efficient transfer of proteins to diazobenzyloxymethyl cellulose is demonstrated.