A brief review of other notable protein detection methods on blots.

Several methods have been used for detecting proteins on membranes. These include the use of quantum dot luminescent labels, oxyblot immunochemical detection, polymer immunocomplexes, "coupled" probing approach, in situ renaturation of proteins for detecting enzyme activities in crude or purified preparations, immunochromatographic assay, western-phosphatase assay, and the use of Congo red dye, a cosmetic color named Alta, Pro-Q Emerald 488 dye, or amine-reactive dye in combination with alkaline phosphatase and horseradish peroxidase-antibody conjugates for the simultaneous trichromatic fluourescence detection of proteins. Several methods have been used to improve the detection of proteins on membranes, including glutaraldehyde treatment of nitrocellulose blots, elimination of keratin artifacts in immunoblots probed with polyclonal antibodies, and the washing of immunoblots with excessive water and manipulation of Tween-20 in wash buffer.

Detection of La/SS-B by western blot using nanogold-tagged antibodies and silver enhancement.

Immunogold staining with silver enhancement is a versatile, sensitive and specific method for immunodetection of diverse protein antigens separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis and transferred to nitrocellulose or polyvinylidene difluoride membranes. "Next-generation" antibodies tagged with nanogold particles have a wide scope of use including but not limited to immunohistochemistry, western blotting, electron microscopy, fluorescent activated cell sorting procedures, and cell isolation and migration studies. Herein, we describe the use of a nanogold-tagged anti-mouse IgG secondary antibody and silver enhancement methodologies coupled with antigen-specific unlabeled primary antibodies for the detection of the La/SS-B autoantigen by western blotting as a useful alternative to chemiluminescent and enzymatic detection methods.

Protein stains to detect antigen on membranes.

Western blotting (protein blotting/electroblotting) is the gold standard in the analysis of complex protein mixtures. Electroblotting drives protein molecules from a polyacrylamide (or less commonly, of an agarose) gel to the surface of a binding membrane, thereby facilitating an increased availability of the sites with affinity for both general and specific protein reagents. The analysis of these complex protein mixtures is achieved by the detection of specific protein bands on a membrane, which in turn is made possible by the visualization of protein bands either by chemical staining or by reaction with an antibody of a conjugated ligand.

Whole and strip nitrocellulose membrane as well as a new-line-immunoblotting of antigen using the chemiluminescence technique.

There are a number of techniques in the scientific world that researchers use to detect specific antigens. One such technique that has provided many advantages over typical immunochemical staining is chemiluminescence. The emission of visible radiation by compounds once exposed to sunlight has been known for centuries and currently is the main principle for chemiluminescence.

A brief review of other notable protein blotting methods.

A plethora of methods have been used for transferring proteins from the gel to the membrane. These include centrifuge blotting, electroblotting of proteins to Teflon tape and membranes for N- and C-terminal sequence analysis, multiple tissue blotting, a two-step transfer of low and high molecular weight proteins, blotting of Coomassie Brilliant Blue (CBB)-stained proteins from polyacrylamide gels to transparencies, acid electroblotting onto activated glass, membrane-array method for the detection of human intestinal bacteria in fecal samples, protein microarray using a new black cellulose nitrate support, electrotransfer using square wave alternating voltage for enhanced protein recovery, polyethylene glycol-mediated significant enhancement of the immunoblotting transfer, parallel protein chemical processing before and during western blot and the molecular scanner concept, electronic western blot of matrix-assisted laser desorption/ionization (MALDI) mass spectrometry-identified polypeptides from parallel processed gel-separated proteins, semidry electroblotting of peptides and proteins from acid-urea polyacrylamide gels, transfer of silver-stained proteins from polyacrylamide gels to polyvinylidene difluoride (PVDF) membranes, and the display of K(+) channel proteins on a solid nitrocellulose support for assaying toxin binding. The quantification of proteins bound to PVDF membranes by elution of CBB, clarification of immunoblots on PVDF for transmission densitometry, gold coating of nonconductive membranes before MALDI tandem mass spectrometric analysis to prevent charging effect for analysis of peptides from PVDF membranes, and a simple method for coating native polysaccharides onto nitrocellulose are some of the methods involving either the manipulation of membranes with transferred proteins or just a passive transfer of antigens to membranes.

Grid-immunoblotting.

Grid-Immunoblotting is a fast, simple, and efficient method for simultaneously testing multiple allergens utilizing small amount of antibody.

Ultrarapid electrophoretic transfer of high and low molecular weight proteins using heat.

An ultrarapid method for the electrophoretic transfer of high and low molecular weight proteins to nitrocellulose membranes following sodium dodecyl sulfate (SDS) polyacrylamide gel is described here. The transfer was performed with heated (70-75 degrees C) normal transfer buffer from which methanol had been omitted. Complete transfer of high and low molecular weight antigens (molecular weight protein standards, a purified protein, and proteins from a human tissue extract) could be carried out in 10 min for a 7% (0.

A miniaturized blotting system for simultaneous detecting of different autoantibodies.

Sera of tumor patients frequently contain autoantibodies to tumor-associated antigens. Here we describe a miniaturized immunoblot platform allowing us to screen sera of patients for the presence of autoantibodies to ten autoantigens in parallel.

Nonelectrophoretic bidirectional transfer of a single SDS-PAGE gel with multiple antigens to obtain 12 immunoblots.

Protein blotting is an invaluable technique in immunology to detect and characterize proteins of low abundance. Proteins resolved on sodium dodecyl sulfate (SDS) polyacrylamide gels are normally transferred electrophoretically to adsorbent membranes such as nitrocellulose or polyvinylidene diflouride membranes. Here, we describe the nonelectrophroretic transfer of the Ro 60 (or SSA) autoantigen, 220- and 240-kD spectrin antigens, and prestained molecular weight standards from SDS polyacrylamide gels to obtain up to 12 immunoblots from a single gel and multiple sera.

Affinity immunoblotting for analysis of antibody clonotype distribution in a lupus patient developing anti-Ro 60 over time.

We describe a sensitive and specific method to analyze specific antibody clonotype changes in a patient with systemic lupus erythematosus who developed autoantibodies to the Ro 60 autoantigen under observation. Patient sera collected over several years were separated by flatbed isoelectric focusing and analyzed by affinity immunoblotting utilizing Ro 60-coated nitrocellulose membrane. When the Ro 60-coated nitrocellulose was laid over the surface of the IEF gel, the antibodies present on the surface of the acrylamide gel bound the Ro antigen on the nitrocellulose.

Tryptic peptide purification using polyvinylidene difluoride membrane for matrix-assisted laser desorption ionization time of flight mass spectrometry.

Matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI TOF MS) is extremely sensitive to minor impurities in tryptic peptide digests, resulting in suppression of the signal obtained. Therefore, it becomes necessary to purify the sample, especially those samples that fail to yield good mass spectra. Here, we describe a simple protocol using polyvinylidene difluoride (PVDF) membrane for purifying tryptic peptides prior to mass spectrometric analysis.

Introduction to protein blotting.

Protein blotting is a powerful and important procedure for the immunodetection of proteins following electrophoresis, particularly proteins that are of low abundance. Since the inception of the protocol for protein transfer from an electrophoresed gel to a membrane in 1979, protein blotting has evolved greatly. The scientific community is now confronted with a variety of ways and means to carry out this transfer.

Systemic lupus erythematosus and C1q: A quantitative ELISA for determining C1q levels in serum.

C1q is of interest in systemic lupus erythematosus (SLE) research due to deficiencies in its activity being associated with the disease. Current published protocols for measuring C1q vary greatly in their results and ease of reproducibility. Due to this, average C1q concentrations have been reported between 56 and 276 microg/mL in non-SLE serum.

Replication of the BANK1 genetic association with systemic lupus erythematosus in a European-derived population.

Systemic lupus erythematosus (SLE) is an autoimmune disease with highly variable clinical presentation. Patients suffer from immunological abnormalities that target T-cell, B-cell and accessory cell functions. B cells are hyperactive in SLE patients.

Molecular evidence for the identity of the Magenta petrel.

A lone petrel was shot from the decks of an Italian warship (the 'Magenta') while it was sailing the South Pacific Ocean in 1867, far from land. The species, unknown to science, was named the 'Magenta petrel' (Procellariiformes, Procellariidae, Pterodroma magentae). No other specimens of this bird were collected and the species it represented remained a complete enigma for over 100 years.

Ancient genetic variation in one of the world's rarest seabirds.

The Chatham Island Taiko (Tchaik, Pterodroma magentae) is one of the world's rarest seabirds. In the past there were millions of breeding pairs of Taiko and it was the most abundant burrowing petrel on Chatham Island. The present population consists of just 120-150 birds, including only 8-15 breeding pairs.

Chronic lymphocytic leukemia cells recognize conserved epitopes associated with apoptosis and oxidation.

Chronic lymphocytic leukemia (CLL) represents the outgrowth of a CD5(+) B cell. Its etiology is unknown. The structure of membrane Ig on CLL cells of unrelated patients can be remarkably similar.

Inhalation of environmental stressors & chronic inflammation: autoimmunity and neurodegeneration.

Human life expectancy and welfare has decreased because of the increase in environmental stressors in the air. An environmental stressor is a natural or human-made component present in our environment that upon reaching an organic system produces a coordinated response. This response usually involves a modification of the metabolism and physiology of the system.

Klinefelter's syndrome (47,XXY) in male systemic lupus erythematosus patients: support for the notion of a gene-dose effect from the X chromosome.

Objective: Systemic lupus erythematosus (SLE) is a systemic autoimmune disease that predominantly affects women. Despite isolated reports of patients with coexisting Klinefelter's syndrome (47,XXY) and SLE, no association of Klinefelter's syndrome with SLE or any other autoimmune disease has been established. The present study was undertaken to investigate the prevalence of Klinefelter's syndrome in a large population of patients with SLE.