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An innovative integrated paper-based microdevice was developed for protein separation by isoelectric focusing (IEF), allowing for robust design thanks to a 3D-printed holder integrating separation channel, reservoirs, and electrodes. To reach robustness and precision, the optimization focused on the holder geometry, the paper nature, the reservoir design, the IEF medium, and various focusing parameters. A well-established and stable pH gradient was obtained on a glass-fiber paper substrate with simple sponge reservoirs, and the integration of the electrodes in the holder led to a straightforward system. The separation medium composed of water/glycerol (85/15, v/v) allowed for reducing medium evaporation while being an efficient medium for most hydrophobic and hydrophilic proteins, compatible with mass spectrometry detection for further proteomics developments. To our knowledge, this is the first report of the use of glycerol solutions as a separation medium in a paper-based microdevice. Analytical performances regarding pH gradient generation, pI determination, separation efficiency, and resolution were estimated while varying the IEF experimental parameters. The overall process led to an efficient separation within 25 min. Then, this methodology was applied to a sample composed of saliva doped with proteins. A minimal matrix effect was evidenced, underscoring the practical viability of our platform. This low-cost, versatile and robust paper-based IEF microdevice opens the way to various applications, ranging from sample pre-treatment to integration in an overall proteomic-on-a-chip device., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Integration and storage of bioactive reagents is an important and challenging task in microfluidic paper-based analytical devices (μPADs). Here, we developed a convenient and universal method to store proteins and preserve their activities in μPADs by using aqueous two-phase systems (ATPs) evolved film. A polyethylene glycol (PEG)-dextran (DEX) double-layer film was formed through dehydration of ATPs. Functional biomolecules were stored in the bottom DEX layer on the basis of the biased partitioning and rehydrated conveniently by simple addition of buffer solution at usage. As a demonstration, enzyme immunoassay (EIA) of carcinoembryonic antigen was performed successfully on μPAD integrated with antibodies. Even after 104 days of storage at 4 °C and ambient conditions, the EIA signal just lost less than 10% and 30%, which meet the storage requirements of invitro diagnosis reagents. The ATPs evolved double-layer film has double functions of stabilization and insulation, and provide a high efficiency of biomolecule preservation, thereby promoting the applications of μPADs in POC diagnostic assay., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022 Elsevier B.V. All rights reserved.)

Proteinuria is a condition in which an excessive amount of protein is excreted in urine. It is, among others, an indicator of kidney disease or risk of cardiovascular disease. Rapid and reliable diagnosis and monitoring of proteinuria is of great importance for both patients and their physicians. For that reason, a paper-based sensor for proteinuria diagnosis was designed, optimized, and validated utilizing smartphone-assisted signal acquisition. In the first step, a few commonly employed protein assays were optimized and compared in terms of analytical performance on paper matrix. The tetrabromophenol blue method was selected as the one providing a sufficiently low limit of detection (39 mg·L -1 ) on the one hand and appropriate long-term stability (up to 3 months) on the other hand. The optimized assay was employed for protein-to-creatinine ratio (PCR) determination on a single paper-based sensor. For both analytes the linear ranges were within the clinically relevant range. The analytical usefulness of the developed sensors was demonstrated by a PCR recovery study in artificial urine. The obtained PCR recoveries were from ca. 80 to 150%.

Proteinaceous toxins are harmful proteins derived from plants, bacteria, and other natural sources. They pose a risk to human health due to infection and also as possible biological warfare agents. Paper spray mass spectrometry (PS-MS) with wipe sampling was used to detect proteins from surfaces as a potential tool for identifying the presence of these toxins. Proteins ranging in mass between 12.4 and 66.5 kDa were tested, including a biological toxin simulant/vaccine for Staphylococcal enterotoxin B (SEBv). Various substrates were tested for these representative proteins, including a laboratory bench, a notebook cover, steel, glass, plant leaf and vinyl flooring. Carbon sputtered porous polyethylene (CSPP) was found to outperform typical chromatography paper used for paper spray, as well as carbon nanotube (CNT)-coated paper and polyethylene (PE), which have been previously shown to be well-suited for protein analysis. Low microgram quantities of the protein toxin simulant and other test proteins were successfully detected with good signal-to-noise from surfaces using a porous wipe. These applications demonstrate that PS-MS can potentially be used for rapid, sample preparation-free detection of proteins and biological warfare agents, which would be beneficial to first responders and warfighters.

Paper Spray Ionization (PSI) is commonly applied for the analysis of small molecules, including drugs, metabolites, and pesticides in biological fluids, due to its high versatility, simplicity, and low costs. In this study, a new setup called Solvent Assisted Paper Spray Ionization (SAPSI), able to increase data acquisition time, signal stability, and repeatability, is proposed to overcome common PSI drawbacks. The setup relies on an integrated solution to provide ionization potential and constant solvent flow to the paper tip. Specifically, the ion source was connected to the instrument fluidics along with the voltage supply systems, ensuring a close control over the ionization conditions. SAPSI was successfully applied for the analysis of different classes of biomolecules: amyloidogenic peptides, proteins, and N-glycans. The prolonged analysis time allowed real-time monitoring of processes taking places on the paper tip, such as amyloid peptides aggregation and disaggregation phenomena. The enhanced signal stability allowed to discriminate protein species characterized by different post translational modifications and adducts with electrophilic compounds, both in aqueous solutions and in biofluids, such as serum and cerebrospinal fluid, without any sample pretreatment. In the next future, application to clinical relevant modifications, could lead to the development of quick and cost-effective diagnostic tools.

A green and facile method is presented for in situ synthesis of a novel photoluminescence-quenching nanopaper with a highly-efficient quenching ability, rapid reaction time and long-term storage. The as-prepared nanopaper is further used to construct an aptasensor platform with high performance, rapidness and robustness.

The present review article reports the most innovative methods to detect proteins in historical and archeological samples as well as to characterize proteins used as binders in artworks. Difficulties to ascribe proteins to a certain animal species are often due to post-translational modifications originated by chemical or microbial deterioration during aging. Combining different techniques such as peptide mass fingerprinting and tandem mass spectrometry can solve some of these problems and also allow discrimination between taxonomically related species like sheep and goat. The most studied proteins in bones and textile samples are osteocalcin, collagen and keratin, whereas egg yolk and white proteins, casein and collagen are the most relevant for binders used in old paintings. With the suitable approaches (immune-based methods, DOT-blot, etc…) it is also possible to obtain in situ characterization or analyze the samples directly in the museum laboratories, with the advantage of avoiding artwork damage and expensive external commitments. Recent cutting-edge strategies allowed detection of proteinaceous infection markers that, for instance, were used to establish the cause of death of old Inca mummies and also proved the presence of Yersinia pestis in old documents dating from the period in 17th century in which the plague ravaged Europe.

Isoelectric focusing plays a critical role in the analysis of complex protein samples. Conventionally, isoelectric focusing is implemented with carrier ampholytes in capillary or immobilized pH gradient gel. In this study, we successfully exhibited a carrier ampholyte-free isoelectric focusing on paper-based analytical device. Proof of the concept was visually demonstrated with color model proteins. Experimental results showed that not only a pH gradient was well established along the open paper fluidic channel as confirmed by pH indicator strip, the pH gradient range could also be tuned by the catholyte or anolyte. Furthermore, the isoelectric focusing fractions from the paper channel can be directly cut and recovered into solutions for post analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis and matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry. This paper-based isoelectric focusing method is fast, cheap, simple and easy to operate, and could potentially be used as a cost-effective protein sample clean-up method for target protein analysis with mass spectrometry., (© 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

A concept integrating sampling and protein digestion is introduced here combining fast and simple fabrication by wax printing on filter paper with trypsin immobilized polymer beads. The paper reactors showed promising results with a high degree of protein digestion within fifty minutes in model protein mixtures as well as in human blood. The model protein mixture was used for the evaluation of performance both with and without a reduction and alkylation step. The paper reactors without reduction and alkylation showed between 46% and 75% protein sequence coverage and between five and 20 high confidence peptides (one and five zero missed cleavage peptides, respectively). Compared to a conventional in-solution approach, the paper reactor showed 10% less protein sequence coverage, 29% fewer high confidence peptides and 19% fewer high confidence peptides with zero missed cleavages. Placement of the protein reduction and alkylation step (before or after protein digestion) was shown to be of low importance. The storage stability of the paper reactors with (six weeks) and without (twelve weeks) tryptic peptides was satisfactory. The ability of the paper reactors to digest complex biological samples was investigated by comparison with human whole blood samples prepared using a conventional dried blood spot (DBS) procedure with overnight digestion in non-targeted analysis. The reactors showed a comparable performance with 75 ± 25 for the protein groups compared to 76 ± 5 for the DBS samples. Additionally, 267 ± 72 and 335 ± 11 unique peptides (high confidence) were identified for on-paper digestion and DBS, respectively.

The design of appropriate diagnostic assays for the point of care requires development of suitable biosensors, detection methods, and diagnostic platforms for sensitive, quantitative detection of biological analytes. Protein targets in particular are especially challenging to detect quantitatively and sensitively due to the lack of amplification strategies akin to nucleic acid amplification. However, recent advances in transducer and biosensor design, new detection labels, and paper-based microfluidics may realize the goal of sensitive, fast, portable, and low-cost protein detection. In this review, we discuss the biochemistry, optics, and engineering advances that may be leveraged to design such a sensitive protein diagnostic assay. The binding kinetics, mechanisms of binding in porous networks, and potential transducers are explained in detail. We discuss the relative merits of various optical detection strategies, potential detection labels, optical readout approaches, and image-processing techniques that are amenable to point-of-care use. To conclude, we present a systematic analysis of potential approaches to enhance the sensitivity of paper-based assays. The assay development framework presented here provides bioassay developers a strategy to methodically enhance the sensitivity and point-of-care suitability of protein diagnostics., (© 2017 Elsevier Inc. All rights reserved.)

Paper-based fluorescence resonance energy transfer assay (FRET) is gaining great interest in detecting macro-biological molecule. It is difficult to achieve conveniently and fast detection for macro-biological molecule. Herein, a graphene oxide (GO)-based paper chip (glass fiber) integrated with fluorescence labeled single-stranded DNA (ssDNA) for fast, inexpensive and direct detection of biological macromolecules (proteins and nucleic acids) has been developed. In this paper, we employed the Cy3/FAM-labeled ssDNA as the reporter and the GO as quencher and the original glass fiber paper as data acquisition substrates. The chip which was designed and fabricated by a cutting machine is a miniature biosensor that monitors fluorescence recovery from resonance energy transfer. The hybridization assays and fluorescence detection were all simplified, and the surface of the chip did not require immobilization or washing. A Nikon Eclipse was employed as excited resource and a commercial digital camera was employed for capturing digital images. This paper-based microfluidics chip has been applied in the detection of proteins and nucleic acids. The biosensing capability meets many potential requirements for disease diagnosis and biological analysis., (Copyright © 2016. Published by Elsevier B.V.)

Conventional microfluidic devices are typically complex and expensive. The devices require the use of pneumatic control systems or highly precise pumps to control the flow in the devices. This work investigates an alternative method using paper based microfluidic devices to replace conventional microfluidic devices. Size based separation and extraction experiments conducted were able to separate free dye from a mixed protein and dye solution. Experimental results showed that pure fluorescein isothiocyanate could be separated from a solution of mixed fluorescein isothiocyanate and fluorescein isothiocyanate labeled bovine serum albumin. The analysis readings obtained from a spectrophotometer clearly show that the extracted tartrazine sample did not contain any amount of Blue-BSA, because its absorbance value was 0.000 measured at a wavelength of 590nm, which correlated to Blue-BSA. These demonstrate that paper based microfluidic devices, which are inexpensive and easy to implement, can potentially replace their conventional counterparts by the use of simple geometry designs and the capillary action. These findings will potentially help in future developments of paper based microfluidic devices., (Copyright © 2015 Elsevier B.V. All rights reserved.)

The passivation of nonspecific protein adsorption to paper is a major barrier to the use of paper as a platform for microfluidic bioassays. Herein we describe a simple, scalable protocol based on adsorption and cross-linking of poly(oligoethylene glycol methacrylate) (POEGMA) derivatives that reduces nonspecific adsorption of a range of proteins to filter paper by at least 1 order of magnitude without significantly changing the fiber morphology or paper macroporosity. A lateral-flow test strip coated with POEGMA facilitates effective protein transport while also confining the colorimetric reporting signal for easier detection, giving improved performance relative to bovine serum albumin (BSA)-blocked paper. Enzyme-linked immunosorbent assays based on POEGMA-coated paper also achieve lower blank values, higher sensitivities, and lower detection limits relative to ones based on paper blocked with BSA or skim milk. We anticipate that POEGMA-coated paper can function as a platform for the design of portable, disposable, and low-cost paper-based biosensors.

We have demonstrated a multistep 2-dimensional paper network immunoassay based on controlled rehydration of patterned, dried reagents. Previous work has shown that signal enhancement improves the limit of detection in 2-dimensional paper network assays, but until now, reagents have only been included as wet or dried in separate conjugate pads placed at the upstream end of the assay device. Wet reagents are not ideal for point-of-care because they must be refrigerated and typically limit automation and require more user steps. Conjugate pads allow drying but do not offer any control of the reagent distribution upon rehydration and can be a source of error when pads do not contact the assay membrane uniformly. Furthermore, each reagent is dried on a separate pad, increasing the fabrication complexity when implementing multistep assays that require several different reagents. Conversely, our novel method allows for consistent, controlled rehydration from patterned reagent storage depots directly within the paper membrane. In this assay demonstration, four separate reagents were patterned in different regions of the assay device: a gold-antibody conjugate used for antigen detection and three different signal enhancement components that must not be mixed until immediately before use. To show the viability of patterning and drying reagents directly onto a paper device for dry reagent storage and subsequent controlled release, we tested this device with the malaria antigen Plasmodium falciparum histidine-rich protein 2 (PfHRP2) as an example of target analyte. In this demonstration, the signal enhancement step increases the visible signal by roughly 3-fold and decreases the analytical limit of detection by 2.75-fold.

The first step in curing a disease is being able to detect the disease effectively. Paper-based microfluidic devices are biodegradable and can make diagnosing diseases cost-effective and easy in almost all environments. We created a three-dimesnional (3D) paper device using wax printing fabrication technique and basic principles of origami. This design allows for a versatile fabrication technique over previously reported patterning of SU-8 photoresist on chromatography paper by employing a readily available wax printer. The design also utilizes multiple colorimetric assays that can accommodate one or more analytes including urine, blood, and saliva. In this case to demonstrate the functionality of the 3D paper-based microfluidic system, a urinalysis of protein and glucose assays is conducted. The amounts of glucose and protein introduced to the device are found to be proportional to the color change of each assay. This color change was quantified by use of Adobe Photoshop. Urine samples from participants with no pre-existing health conditions and one person with diabetes were collected and compared against synthetic urine samples with predetermined glucose and protein levels. Utilizing this method, we were able to confirm that both protein and glucose levels were in fact within healthy ranges for healthy participants. For the participant with diabetes, glucose was found to be above the healthy range while the protein level was in the healthy range.

Carbon nanotube (CNT) has been utilized for the biological detection due to its extremely sensitive to biological molecules. A paper-based CNT sensing microfluidic device has been developed for the detection of protein, i.e., biotin-avidin, binding. We have developed a fabrication method that allows controlled deposition of bundled CNTs with well-defined dimensions to form sensors on paper. Then, polydimethyl siloxane (PDMS) was used to pattern the hydrophobic boundary on paper to form the reaction sites. The proposed fabrication method is based on vacuum filtration process with a metal mask covering on a filter paper for the definition of the dimension of sensor. The length, width, and thickness of the CNT-based sensors are readily controlled by the metal mask and the weight of the CNT powder used during the filtration process, respectively. Homogeneous deposition of CNTs with well-defined dimensions can be achieved. The CNT-based sensor on paper has been demonstrated on the detection of the protein binding. Biotin was first immobilized on the CNT's sidewall and avidin suspended solution was applied to the site. The result of the biotin-avidin binding was measured by the resistance change of the sensor, which is a label-free detection method. It showed the CNT is sensitive to the biological molecules and the proposed paper-based CNT sensing device is a possible candidate for point-of-care biosensors. Thus, electrical bio-assays on paper-based microfluidics can be realized to develop low cost, sensitive, and specific diagnostic devices.

A method for patterning narrow lines of biomolecules onto nitrocellulose membranes using laboratory syringe pumps is described. One syringe pump is used to drive the biomolecule solution through a needle, while another modified syringe pump acts as a one-dimensional translation stage, moving the needle across the membrane much like a pen. This method consumes very small volumes of reagents, and is a viable option for laboratory-scale fabrication and prototyping of point-of-care rapid diagnostic test strips.

Paper spray is developed as a direct sampling ionization method for mass spectrometric analysis of complex mixtures. Ions of analyte are generated by applying a high voltage to a paper triangle wetted with a small volume (<10 microL) of solution. Samples can be preloaded onto the paper, added with the wetting solution, or transferred from surfaces using the paper as a wipe. It is demonstrated that paper spray is applicable to the analysis of a wide variety of compounds, including small organic compounds, peptides, and proteins. Procedures are developed for analysis of dried biofluid spots and applied to therapeutic drug monitoring with whole blood samples and to illicit drug detection in raw urine samples. Limits of detection of 50 ng/mL (or 20 pg absolute) are achieved for atenolol in bovine blood. The combination of sample collection from surfaces and paper spray ionization also enables fast chemical screening at high sensitivity, for example 100 pg of heroin distributed on a surface and agrochemicals on fruit peels are detectable. Online derivatization with a preloaded reagent is demonstrated for analysis of cholesterol in human serum. The combination of paper spray with miniature mass spectrometers offers a powerful impetus to wide application of mass spectrometry in nonlaboratory environments.

Paper has been proposed as an inexpensive and versatile carrier for microfluidics devices with abilities well beyond simple capillary action for pregnancy tests and the like. Unlike standard microfluidics devices, extracting a fluid from the paper is a challenge and a drawback to its broader use. Here, we extract fluid from narrow paper strips using surface acoustic wave (SAW) irradiation that subsequently atomizes the extracted fluid into a monodisperse aerosol for use in mass spectroscopy, medical diagnostics, and drug delivery applications. Two protein molecules, ovalbumin and bovine serum albumin (BSA), have been preserved in paper and then extracted using atomized mist through SAW excitation; protein electrophoresis shows there is less than 1% degradation of either protein molecule in this process. Finally, a solution of live yeast cells was infused into paper, which was subsequently dried for preservation then remoistened to extract the cells via SAW atomization, yielding live cells at the completion of the process. The successful preservation and extraction of fluids, proteins and yeast cells significantly expands the usefulness of paper in microfluidics.

The purpose of this study was to compare the efficiency of protein elution from several types of gingival crevicular fluid (GCF) collection papers when the volume of the inoculated protein and the elution methods were constant. Various concentrations of bovine serum albumin (BSA) and 14C-BSA were placed onto strips of Whatman #1 [W1], Whatman 3 MM chromatographic [W3], Periopaper (ProFlow) [P] and Periopaper (Harco) [H], and recovered proteins measured following a non-optimized centrifugal elution technique. There were significant differences in % recovery of BSA and 14C-BSA from the papers, which was dependent on both the type of paper and the concentration of the inoculated protein; that is, proteins at the lowest concentrations were less efficiently eluted from GCF collection papers than those at higher concentrations. Equations for regression lines of elution efficiency were quadratic. Thus, our data suggest significant differences in the efficiency for elution of BSA from absorbent papers when the volume of the inoculated fluid and the elution technique were constant. Previous variable or conflicting experimental data between research groups may have resulted from incomplete elution of proteins from GCF collection papers, possibly due to entrapment within, or binding of GCF proteins to the paper.

The preparation of homogeneous ultrathin native polyacrylamide gels, using a basic as well as an acidic buffer system is described. The basic buffer system consists of Tris-HC1/Tris-glycine, the same buffer as in sodium dodecyl sulfate (SDS)-gel electrophoresis but without SDS. The acidic system uses potassium acetate, pH 4.3, as gel buffer and beta-alanine, pH 4.6, acetic acid as electrolytes. The gels are covalently bound on glass plates. Binding of acidic gels requires a special pretreatment of glass plates. The whole procedure is simple and extraordinarily fast: 100-120 min from the start of gel preparation to the end of electrophoresis. Coomassie staining is done in 40 min and silver staining in 90 min. The native gels are excellently suited for diffusion blotting. Further attractive properties of these gels are easy handling, simple drying and dimensional stability.

We describe a method by which complex protein mixtures are fractionated by standard one-dimensional Na-DodSO4/polyacrylamide gel electrophoresis or O'Farrell two-dimensional gel electrophoresis and then are efficiently and rapidly transferred electrophoretically to diazobenzyloxymethyl- or diazophenylthioether-paper and analyzed by immunoautoradiography. The method is illustrated with protein extracts of human KB cells infected with adenovirus type 2. Proteins were transferred from gels without decrease in resolution and with an increase in the sensitivity of detection by autoradiography when [35S]-methionine-labeled proteins were used. When unlabeled proteins were transferred, low levels of virus encoded proteins could be detected by sequential treatment of diazobenzyloxymethyl-paper with anti-adenovirus type 2 virion or anti-73,000 DNA binding protein and 125I-labeled Staphyloccus aureus protein A. Covalently bound viral proteins retained immunologic reactivity after dissociation of the protein A and antibody, By one-dimensional gel transfer/immunoautoradiography, seven virion proteins were detected as prominent bands and several others as weaker bands. By two-dimensional gel transfer/immunoautoradiography, several additional viral proteins were detected. By use of anti-DNA binding protein serum, the Mr 73,000 protein and Mr 41,000-48,000 subspecies were detected. A protein present at a concentration of approximately 1 part in 100,000 of the total protein can be identified in cell extracts. This method may be applicable to various biological problems requiring resolution and detection of small amounts of specific proteins that can be recognized immunologically or that can be detected by binding to specific radiolabeled DNA or RNA sequences or hormones.

We examine the effects of filter efficiency on DNA-protein binding data obtained by the popular nitrocellulose filter-binding assay. Graphical procedures for determining the efficiency parameter epsilon (for the efficiency of retention of DNA on the filter, per bound protein molecule) are established. Filter efficiency modified formulas for determining thermodynamic binding parameters are derived for four simple prototypes of DNA-protein binding systems. The effects of experimental error on discrimination between models are considered. Finally, we discuss conceptual errors often made in calculating binding stoichiometry from filter-binding experiments and suggest a general protocol for the analysis of filter-binding data.

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.

Allergenic proteins were extracted from one silk batch that was imported to be used as filling material for bed mattresses and rugs. IgE and IgG antibodies to the extracted silk proteins were measured by RAST in sera of nine silk-sensitive persons as well as in sera of healthy control donors. Silk proteins were fractionated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis into 12 polypeptides of molecular weights between 14 and 70 kilodaltons. By means of the immunoblot technique, IgE and IgG antibodies to the individual silk polypeptides could be detected. Sera of silk-sensitive persons contained high titers of IgE and low titers of IgG antibodies to the separated silk polypeptides. Sera of control donors contained low IgG antibody titers to a limited number of these polypeptides.

We have quantitatively examined the various parameters affecting the electrotransfer and sequence analysis of proteins from sodium dodecyl sulfate (SDS) gels to derivatized glass fiber paper or to polyvinyldifluoride (PVDF) membranes. Transfer yields in the range of 90-95% can be obtained for proteins in the molecular weight range of 10-90 kDa for transfer from 12% SDS gels to glass fiber paper derivatized with either QAPS (N-trimethoxysilylpropyl-N,N,N-trimethylammonium chloride) or APS (aminopropyltriethoxysilane). In order to achieve these yields, it was necessary to modify the conditions described by R. Aebersold et al. (J. Biol. Chem. 261, 4229-4238, 1986). We activated the glass fiber paper with dilute ammonia water and derivatized the activated glass fiber paper with QAPS and APS in anhydrous solvents which were allowed to slowly absorb moisture during the derivatization process. The transfer yield varied with transfer time versus molecular weight of the protein for a given percentage gel. Shorter transfer times and higher yields were obtained for higher molecular weight proteins on 8% gels. Lower molecular weight protein gave higher yields from 12% gels under similar transfer conditions. Sequencing yields of the transferred proteins were in the range of 40-80%, but a number of background peaks were observed on HPLC analysis of the phenylthiohydantoin amino acid derivatives. Transfer yields in the range of 85-95% were observed for similar experiments with PVDF membranes. In order to achieve these yields, it was necessary to modify the conditions described by P. Matsudaira (J. Biol. Chem. 262, 10035-10038, 1987). A lower voltage and longer transfer times gave higher transfer yields. In order to achieve consistently high transfer yields, it was also necessary to precoat the PVDF membranes with Polybrene. The PVDF membranes were cut into approximately 1-mm-wide strips and inserted into a continuous flow reactor (J. E. Shively, P. Miller, and M. Ronk, Anal. Biochem. 163, 517-525, 1987) for sequence analysis. Overall yields of samples loaded onto gels, electrotransferred to Polybrene-coated PVDF membranes, and sequenced ranged from 50-60% for beta-lactoglobin (10-50 pmol loaded onto SDS gels) to 20-30% for bovine serum albumin and soybean trypsin inhibitor (50 pmol loaded onto SDS gels). A comparison of the two methods shows clear advantages for the PVDF membranes over the derivatized glass fiber paper, including the ability to directly sequence the Coomassie blue-stained PVDF membranes, and the lower backgrounds observed on subsequent sequence analysis.

The detectability by India ink staining of proteins and peptides dot-blotted on nitrocellulose paper was assessed before and after fixation. Fixation considerably increased the detectability of proteins and peptides. Denaturation by KOH treatment or baking at 100 degrees C for 15 min gave the best results. Precipitation by isopropanol/acetic acid gave intermediate results, whereas crosslinking with glutaraldehyde improved the detectability of small peptides, but not of proteins. Ferridye and Aurodye were also tested after baking. Both dyes were more sensitive and stained more proteins and peptides than India ink. In all cases the detectability of peptides smaller than Mr 1500 was poor.

A simple method for covalent coupling of proteins to filter paper modified with quinone groups is described. This paper, termed 'Key paper' is flexible, stable on storage and does not require any activation before use. Proteins bound to Key paper can be detected by enzyme immunoassay, radioimmunoassay or Coomassie blue staining. Bound enzymes retain their enzymatic activity. Nucleic acids do not bind and do not interfere with the activity of the bound proteins. Because of its mechanical and chemical properties Key paper is a good matrix for electroblotting and for direct in situ analysis of proteins.

Link protein (Mr = 42,000) is an integral component of cartilage as well as of some noncartilagenous tissues. In cartilage, it forms a macromolecular complex with cartilage proteoglycan and hyaluronic acid, but its function in other tissues is unknown. We provide evidence here that the link protein of cartilage binds well to native collagen types I and III. The binding occurs only if both link protein and collagen are native. The binding of link protein to collagen type fibrils is higher than to monomeric collagen. Link protein binding to collagen fibrils is saturable and occurs at molar ratio of collagen to link protein of 7-13:1. These data suggest that the link protein binds to collagen and that the binding requires the collagen to be in its native triple helical structure. This interaction may play a role in collagen fibril formation.

India ink staining of proteins that have been electrotransfer blotted onto nitrocellulose paper is described. This stain proved to be a useful adjunct to the enzyme-linked immunoelectrotransfer blot technique. It is more sensitive than Coomassie blue, amido black, and fast green stains and is simple to use.

Recent studies have demonstrated that antibodies from about half of patients with pemphigus foliaceus (PF) bind to a 160 kd polypeptide ("PF antigen") in sodium dodecyl sulfate (SDS) extracts of normal human epidermis. Desmoglein (DG) I, a glycoprotein enriched in desmosomal cores, is approximately the same m.w. as PF antigen. To demonstrate that PF autoantibodies bind to DG I, we used a monoclonal IgG antibody (MmDGI-1) that was raised against bovine muzzle desmosomal cores, and that specifically binds DG I. Double immunofluorescence labeling was performed on the same section of normal human skin with PF antibodies, detected by fluorescein-conjugated goat anti-human IgG, and MmDGI-1, detected by rhodamine-conjugated goat anti-mouse IgG. The pattern of reactivity with both antibodies was identical. Immunoblotting studies on proteins extracted from normal human epidermis and separated by SDS-polyacrylamide gel electrophoresis demonstrated that PF antibodies and MmDGI-1 bound co-migrating polypeptide bands of approximate m.w. 160,000. To confirm that these were identical polypeptides, we performed immunoblots of these epidermal extracts that were separated by two-dimensional gel electrophoreses (isoelectric focusing followed by SDS-PAGE). PF antibodies and MmDGI-1 bound identical spots with pI approximately 5.4 to 5.7 and m.w. approximately 160,000. These studies demonstrate that autoantibodies from certain patients with PF, a disorder of cell adhesion, bind to DG I, a desmosomal core glycoprotein.