Two-Dimensional Gel Electrophoresis: Vertical Isoelectric Focusing.

Two-dimensional gel electrophoresis is one of the most powerful tools for separating proteins based on their size and charge. Two-dimensional gel electrophoresis (2-DE) is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

Curcumin/Turmeric as an Environment-Friendly Protein Gel Stain.

Curcumin, the main curcuminoid in food spice turmeric, is insoluble in water at room temperature. We showed that curcumin can be solubilized in water with the application of heat (100 °C). Here we demonstrate that heat-solubilized curcumin can serve as a nontoxic and environment-friendly fluorescent/colorimetric reversible protein stain. Curcumin, the yellow pigment found in the rhizomes of the perennial herb Curcuma longa (turmeric), is insoluble in aqueous solvents. However, heat solubilization in water renders 1.5% of curcumin soluble. Curcumin solubilized by ethanol or alkali is ineffective in staining proteins. Heat-solubilized curry spice turmeric also stains proteins. Staining is achieved in 30 min, with a sensitivity almost equaling that of Coomassie Brilliant Blue (CBB). Destaining is not required and excess curcumin/turmeric can be discarded into the sink. Binding of proteins by silver inhibits curcumin binding, suggesting similarity of protein binding by silver and curcumin. It costs $1.5-2.0 to stain a mini-gel with curcumin, while turmeric costs less than 0.005 cent. CBB staining/destaining costs about two cents. Curcumin/turmeric, thus, can serve as an ideal nontoxic protein stain.

Paper Adsorbents Remove Coomassie Blue from Gel Destain and Used Gel Stain in an Environment-Friendly Manner.

Coomassie Brilliant Blue (CBB), used to stain protein gels, is known to be toxic. Therefore, laboratories do not discard used CBB into the sink owing to the possibility of it contaminating drinking water supplies. We tested the ability of various paper adsorbents to adsorb CBB released from gels during destaining. The efficiency was as follows-Kimwipes > Teri towels > multifold towels > Whatman numbers 1 and 3 filter papers. Addition of three Kimwipes during destaining helped adsorb the dye released from a CBB-stained mini-gel. Stain removal with Kimwipes helps reduce destain use, organic waste accumulation, enable recycling of nonradioactive destaining solution and is 7.5-fold cheaper than an available method for CBB disposal. Next, we used Kimwipes to deplete the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. Seventy five Kimwipes successfully helped remove the dye from a 0.05% CBB staining solution in 5-10 min. The blue Kimwipes did not release the CBB stain even when squeezed dry after incubation in various salts, water, or acid solutions for 5 weeks. The CBB removed thus can be simply disposed of as solid waste and will not leach out from solid landfills. Kimwipes, thus, enables CBB disposal in an environmentally friendly manner and allows for recycling of destaining solution.

Immunoblotting of Antigens: Whole, Strip, and New-Line Nitrocellulose Membrane Immunoblotting Using the Chemiluminescence Technique.

Antigen detection is a well-known tool in the scientific world that is used by clinicians and researchers to detect specific antigens in diagnosing diseases or for other medical/environmental discoveries. Antigen detection is introduced in various forms over the past decades. These techniques are often evaluated by their sensitivity, accuracy, and ease of use. One technique that has provided many advantages over typical immunochemical staining is the use of chemiluminescence. This technique has been used in various scientific fields, anywhere from clinical diagnosis to environmental research. The emission of visible radiation by compounds once exposed to sunlight has been known for centuries and currently is the main principle for chemiluminescence. Here, we introduce three different chemiluminescence techniques that are widely used in immunodetection of antigens: (a) whole membrane chemiluminescence detection, (b) strip membrane chemiluminescence detection, and

A miniaturized blotting system for simultaneous detection 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.

Induction of anti-Ro60/anti-La by immunisation with spectrin and induction of anti-spectrin by immunisation with Ro60 and 4-hydroxy-2-nonenal-modified Ro60 immunisation.

OBJECTIVES: The Ro ribonucleoprotein particle, targeted in systemic lupus erythematosus (SLE) and Sjögren's syndrome (SS), includes Ro60 (SSA) and La (SSA) autoantigens. Anti-Ro60 occurs in SLE and SS. The importance of α-fodrin and spectrin as well as anti-Ro and anti-fodrin/spectrin antibodies in SS and SLE, led us to hypothesise that rabbit immunisation with Ro60 or 4-hydroxy-2-nonenal-modified Ro60 would induce anti-spectrin. In addition, we hypothesised that antibodies to Ro60 and La will develop in animals immunised with spectrin. METHODS: Two NZW rabbits each were immunised with 4-hydroxy-2-nonenal-modified Ro60 or unmodified Ro60. Methods used included ELISA, including an inside-out RBC membrane ELISA, and Crithidia lucilae assays. RESULTS: Commercial anti-spectrin sera bound significantly to Ro60 (OD 2.6 ± 0.1), Ro60 multiple antigenic peptides (MAPs) (3 out of 21 Ro60 MAPs), La (OD 4.4±0.5), and La fragments as well as to double stranded DNA but not to BSA (OD 0.6±0.1). Anti-spectrin binding to purified spectrin could be inhibited by spectrin (>95%), and Ro60 or La (70%). When the binding of anti-spectrin was tested against a nested set of La fragments we found that a N4 fragment representing the C-terminal 250 aa (aa 159 to 408) bound the strongest (OD=4.12) followed by a N9 fragment (the C-terminal 36aa; aa373 to 408 (OD=1.36). Also, significant anti-spectrin antibody levels were induced by Ro60 and HNE-modified Ro60 immunisation. CONCLUSIONS: We found intermolecular epitope spreading from Ro60/La to spectrin and vice versa, and this may have pathological significance in these animal models of autoimmunity.

Two-dimensional gel electrophoresis: vertical isoelectric focusing.

Two-dimensional gel electrophoresis (2-DE) is one of the most powerful tools for separating proteins based on their size and charge. 2-DE is very useful to separate two proteins with identical molecular weights but different charges, which cannot be achieved with just sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Here, a simpler and easier version of 2-DE is presented which is also faster than all the currently available techniques. In this modified version of 2-DE, isoelectric focusing is carried out in the first dimension using a vertical SDS-PAGE apparatus. Following the first-dimensional IEF, each individual lane is excised from the IEF gel and, after a 90° rotation, is inserted into a second-dimensional SDS-PAGE, which can be stained with Coomassie Brilliant Blue for protein analysis or immunoblotted for further analysis. This version of IEF can be run in less than 2 h compared to the overnight run required by O'Farrell's method. Difficult tube gel casting and gel extrusion as well as tube gel distortion are eliminated in our method. This method is simpler, faster, and inexpensive. Both dimensions can be done on the same SDS-PAGE apparatus, and up to ten samples can be run simultaneously using one gel.

Spicy SDS-PAGE gels: curcumin/turmeric as an environment-friendly protein stain.

Gel proteins are commonly stained with calorimetric/fluorescent dyes. Here, we demonstrate that heat-solubilized curcumin can serve as a nontoxic and environment-friendly fluorescent/colorimetric reversible protein stain. Curcumin, the yellow pigment found in the rhizomes of the perennial herb Curcuma longa (turmeric), is insoluble in aqueous solvents. However, heat (100°C) solubilization in water renders 1.5% of curcumin soluble. Curcumin solubilized by ethanol or alkali is ineffective in staining proteins. Heat solubilized curry spice turmeric stains proteins similarly. Staining is achieved in 30 min, with a sensitivity almost equaling that of Coomassie Brilliant Blue (CBB). Destaining is not required, and excess curcumin/turmeric can be discarded into the sink. Binding of proteins by silver inhibits curcumin binding, suggesting similarity of protein binding by silver and curcumin. It costs $1.5-2.0 to stain a mini-gel with curcumin, while turmeric costs less than 0.005 cent. CBB staining/destaining costs about 2 cents. However, CBB is toxic and its use necessitates specialized disposal efforts. Curcumin/turmeric, thus, can serve as an ideal nontoxic protein stain.

Coomassie Brilliant Blue removal/disposal from gel destain and used gel stain in an environment-friendly manner.

Toxic reagents are employed to destain Coomassie Brilliant Blue (CBB) stained gels. We tested the efficacy of various paper adsorbents in adsorbing CBB released from gels during destaining. Kimwipes were the most efficient, followed by Teri towels, multifold towels, and Whatman (numbers 1 and 3) filter papers. Three Kimwipes added during destaining of a CBB-stained mini-gel helped adsorb the released dye. Thus, stain removal with Kimwipes helps reduce destain use and organic waste accumulation, enables recycling of nonradioactive destaining solution, and is 7.5-fold cheaper than an available method for CBB disposal. Next, we used Kimwipes to deplete the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. Seventy-five Kimwipes successfully helped remove the dye from a 0.05% CBB staining solution in 5 to 10 min. The blue-colored Kimwipes did not release the stain even when squeezed dry after incubation in various salts, water, or acid solutions for five weeks. The CBB removed thus can be simply disposed as solid waste and will not leach out from solid landfills. Kimwipes, thus, enables CBB disposal in an environmentally friendly manner and allows recycling of destaining solution.

An overview of Western blotting for determining antibody specificities for immunohistochemistry.

Despite its overall simplicity, protein blotting or Western blotting has been proven to be a powerful procedure for the immunodetection of proteins, especially those that are of low abundance, following electrophoresis. The usefulness of this procedure stems from its ability to provide simultaneous resolution of multiple immunogenic antigens within a sample for detection by specific antibodies. Protein blotting has evolved greatly since its inception and researchers have a variety of ways and means to carry out this transfer. This procedure is used in combination with other important antibody-based detection methods such as enzyme-linked immunosorbant assay and immunohistochemistry to provide confirmation of results both in research and diagnostic testing. Specificity of antibodies used for immunohistochemistry is of critical importance and therefore Western blot is a "must" to address antibodies' specificity.

Degree of modification of Ro60 by the lipid peroxidation by-product 4-hydroxy-2-nonenal may differentially induce Sjögren syndrome or systemic lupus erythematosus in BALB/c mice.

Our previous work showed that immunization of rabbits with 4-hydroxy-2-nonenal-modified Ro60 (HNE-Ro60) accelerates autoimmunity. We extended this model into mice, hypothesizing that the severity of autoimmunity would be dependent on the degree of HNE modification of Ro60. Five groups of BALB/c mice (10/group) were used. Group I was immunized with Ro60. Groups II to IV were immunized with Ro60 modified with 0.4 mM (low), 2 mM (medium), and 10 mM (high) HNE, respectively. Group V controls received Freund's adjuvant. A rapid abrogation of tolerance to Ro60/La antigens occurred in mice immunized with HNE-modified Ro60, especially in the low and medium HNE-Ro60 groups. Lymphocytic infiltration and significantly high decrement in salivary flow (37%) compared to controls was observed only in the high HNE-Ro60 group, suggesting induction of a Sjögren syndrome-like condition in this group. Anti-dsDNA occurred only in mice immunized with medium HNE-Ro60. This group did not have a significant decrement in salivary flow, suggesting induction of a systemic lupus erythematosus-like manifestation in this group. Significantly high antibodies to Ro60 were found in saliva of mice in the low and medium HNE-Ro60 and the Ro60 groups, as well as anti-HNE Ro60 in the low and medium HNE-Ro60 groups. Understanding the mechanism of this differential induction may help discriminate between these two autoimmune diseases.

Environmentally safe removal/disposal of Coomassie Brilliant Blue from gel destain and used gel stain.

Gel destaining following Coomassie Brilliant Blue (CBB) staining involves the use of toxic reagents. Here we demonstrate the efficacy of various paper adsorbents in adsorbing CBB. Kimwipes adsorbed the best, followed by Teri towels, multifold towels, and Whatman numbers 1 and 3 filter papers. Three Kimwipes completely adsorbed the dye released from a CBB-stained mini-gel. Nonradioactive destain solution can, therefore, be recycled for destaining CBB-stained gels. Stain removal with Kimwipes helps in reducing destain use and in reducing organic liquid waste, and it is 7.5-fold cheaper compared with an available method for CBB disposal. Following this, we determined the suitability of this procedure to remove the dye from a used CBB staining solution awaiting proper disposal by our Institutional Safety Office. The dye from a 0.05% CBB staining solution could be removed in 5 to 10 min using 75 Kimwipes. The CBB-adsorbed Kimwipes did not release the stain when squeezed dry even after incubation in various salts over 1week and in water for 5 weeks. The CBB removed allows its easy disposal as solid waste and will not leach out from solid landfills. Thus, stain removal with Kimwipes helps in disposing CBB in an environmentally friendly manner and allows recycling of destaining solution.

Problems with multiple use of transfer buffer in protein electrophoretic transfer.

Two-dimensional gel electrophoresis (2DE) and SDS-PAGE are the two most useful methods in protein separation. Proteins separated by 2DE or SDS-PAGE are usually transferred to membranes using a variety of methods, such as electrophoretic transfer, heat-mediated transfer, or nonelectrophoretic transfer, for specific protein detection and/or analysis. In a recent study, Pettegrew et al. claim to reuse transfer buffer containing methanol for at least five times for transferring proteins from SDS-PAGE to polyvinylidene difluoride. They add 150-200 ml fresh transfer solution each time for extended use as a result of loss of transfer buffer. Finally, they test efficiency of each protein transfer by chemiluminescence detection. Here, we comment on this report, as we believe this method is not accurate and useful for protein analysis, and it can cause background binding as well as inaccurate protein analysis.

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.

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. Here, we introduce three different chemiluminescence techniques that are widely used in immunodetection of antigens: (a) whole membrane chemiluminescence detection, (b) strip membrane chemiluminescence detection, and (c) new line blotting chemiluminescence.

Awaken olfactory receptors of humans and experimental animals by coffee odourants to induce appetite.

Smell and its mechanism has been of interest to scientists for many years. Smell, not only provides a sensual pleasure of food and perfumes for humans but also reminds us of past memories, thoughts, locations and finally warns of dangers such as fire. One of the uses of coffee beans is on perfume counters, enabling people to distinguish between perfume fragrances. We hypothesize that coffee can be also used to refresh olfactory receptors after cooking, since people usually experience loss of appetite after cooking. We have experienced an increase in appetite, after cooking, by smelling coffee beans. This is probably due to the detachment of food odourants from olfactory receptors by the coffee odourant molecules. We also think that coffee smell could be used in animal research studies, to keep animals healthy by stimulating their appetite. In a recent study, 28 different odourants have been identified from coffee. One or more of these odourants may have strong binding affinity to olfactory receptors which results in detachment of other odourants from the receptors. The high vibration intensity from coffee odourant molecules may cause the detachment of food odourant from olfactory receptors. Another hypothesis might be the unique structure of these coffee odourants. Studies need to be done to investigate the effect of coffee smell on salivary flow and appetite in animals and humans.

Four men in a space station - To say nothing of the cow! The quest for finding respite and work in the ultimate frontier.

Fed up with life on earth, four scientists attempt to make it to space to live in the International Space Station (ISS) and carry out experiments. The difficulties in getting selected by NASA, the rigourous training to fly and the risks of the journey to life and health are the rate limiting steps in their quest. They propose commercialization of space and also ferrying cows to space for food as well as generation of biogas. The anaerobic environment is particularly suitable for biogas generation and if successful they plan to get NASA to launch space vehicles to Mars using this natural fuel with the ISS as the staging area.