Gel Staining Research Articles

Features of proteomic profi le of blood serum of children with bronchial asthma who underwent perinatal central nervous system damage

Objective: proteomic analysis of blood serum of children with bronchial asthma with a history of indication of the pathology of the perinatal period.Materials and methods: a comprehensive examination of children suffering from asthma and patients of the control group using proteomic analysis of blood serum was carried out. Proteomic analysis of blood serum was performed using two-dimensional high-resolution gel electrophoresis followed by staining of gels with fl uorescent dye and time-of-fl ight MALDI mass spectrometry. The proteins were identifi ed using Mascot MS peptide fi ngerprint analysis algorithm and Swiss-Prot protein database.Results: comparative analysis of the proteomic profi le of blood serum of children with pathology of the central nervous system in the perinatal period and developed in the subsequent bronchial asthma and control group patients allowed to establish that number proteins products were reduced in this disease. The proteins in the molecular mass range 16-33 kDa (p<0.05) were identifi ed: glutathione peroxidase 3, transthyretin and system components compliment С4В and C3.Conclusion: modifi cation of the production of these proteins indicates the presence of damage at the molecular level, leading to metabolic and functional disorders in the body of a patient with asthma having a history of indicating the pathology of the perinatal period.

Semi-quantitative, spatially resolved analysis of protein deposit layers on membrane surfaces

Fouling distinctly reduces the filtration performance of membranes. A characterization of the fouling in membranes, however, is difficult due to its spatial distribution. Currently applied methods for deposit layer analysis are rather complex or do not offer a spatial resolution. Knowledge of the spatial distribution, however, could be used to improve the design of membranes, modules, and spacers. Staining with Coomassie Brilliant Blue, related to the staining of PAGE gels, is a simple method to visualize and analyze the deposited proteins semi-quantitatively. We improved an existing staining technique for protein deposits on membranes by adding a calibration for the semi-quantitative analysis and optimizing the sample handling. The method provides a spatially resolved analysis of deposited proteins up to a concentration of 10 g m−2. Apart from staining, data processing is described in order to generate false colors or topographic images of deposits. Thus, the paper describes a simple method to assess and visualize the influence of module characteristics such as spacer design on the spatially resolved protein fouling of polymeric and ceramic membranes. Therefore, the method can contribute to the improvement of the module design and processing conditions with regard to the filtration performance.•Visualization of proteinaceous deposits on membranes•Spatially resolved quantification of proteinaceous deposits

Study of gentamicin deposition in cellulose with albumin

Methods of binding antibacterial drugs to the surface of cellulose without the use of oxidizing agents to prevent the occurrence of wound infections have been studied. The immobilization of gentamicin in the complex of partially denatured albumin in the composition with bacterial cellulose has been analyzed. The study was carried out on samples of cellulose synthesized by Gluconacetobacter hansenii. Albumin served as a binding agent, which was used to impregnate cellulose samples, which were then denatured. Using PCR amplifi cation CFX (BioRad), the optimal denaturation temperature was selected. The effectiveness of the immobilization of albumin in the thickness of the cellulose was assessed by staining it with the luminescent dye SYPRO® Ruby Protein Gel Stain, followed by transilluminator detection. Bacterial cellulose impregnated with undenatured albumin was used as a control. Albumin immobilization in bacterial cellulose was observed at temperatures of 65– 95 °C. The antibacterial activity of the complex “cellulose + albumin + gentamicin” was evaluated using a test strain of bacteria Staphylococcus aureus ATCC 25923. The growth inhibition of the test strain of bacteria was observed in all tests with bacterial cellulose in combination with partially denatured albumin and gentamicin. In control samples, in which gentamicin was not immobilized as part of partially denatured albumin, growth inhibition zones of Staphylococcus aureus ATCC 25923 were not noted. It was concluded that by partial denaturation of albumin it is possible to delay antibacterial drugs in the thickness of bacterial cellulose for their further release. A new version of the material suitable for the production of implants and bandages based on bacterial cellulose gel with antibacterial properties is proposed. Dressings based on a composite of bacterial cellulose, albumin and gentamicin are most relevant for the treatment of burns. The presence of gentamicin in their composition is also relevant for the prevention of bacterial infections.

Staining of RNA and DNA on electrophoretic gels and in cytology with juice of Vaccinium myrtillus berries

BackgroundMy early results of cytological chromosome staining with berry juice of blueberry or bilberry (Vaccinium myrtillus) was re-evaluated with staining of electrophoretic agarose and polyacrylamide gels, fractionating DNA, RNA, and proteins. ResultsElectrophoretic gels were stained with juice from berries of V. myrtillus, only filtered, or the diluted filtrate was mixed with acetic acid and 2-propanol. The staining starts in 2 min, with the highest intensity over the background usually appearing in about 30 min. The berry juice stains RNA, DNA, and an unidentified contaminant of molar mass 200–700 g. After differentiation of the gel background, the stained zones appear purple or black. The berry juice staining with or without acetic 2-propanol shows sharp RNA and DNA zones on gels in Vis (visible) light, and the berry juice displaces a preceding staining with ethidium bromide. A secondary staining with ethidium bromide is not able to displace the berry juice in nucleic acids. Cytological staining of sectioned mature barley (Hordeum vulgare) grains with Vaccinium myrtillus juice followed by differentiation in dilute acetic acid shows nuclei and apparent RNA storing compartments in the aleurone cells and stains suberin-containing chalazal cells of the grain crease deeply red, which keeps long. The anthocyanins faintly stain some proteins on gels. The preparation of polymers from cytochrome c and myoglobin was described. ConclusionsThe staining molecules of V. myrtillus anthocyanins apparently intercalate in the nucleic acids competing for the sites, which could be intercalated by ethidium bromide. The juice is suggested to have use in the exchange of intercalating toxic molecules from DNA and RNA, in the inactivation of viruses, and phytotherapy. The juice may have a use as a nontoxic stain in cytology.

Nitric Oxide Donor NOC-18-Induced Changes of Mitochondrial Phosphoproteome in Rat Cardiac Ischemia Model.

Background and objective: Nitric oxide (NO) is known to exert cardioprotective effects against heart ischemic damage and may be involved in ischemic pre- and postconditioning. NO-triggered cardioprotective mechanisms are not well understood but may involve regulation of mitochondrial permeability transition pore (mPTP). In this study, we aimed to identify differentially phosphorylated mitochondrial proteins possibly involved in the NO/protein kinase G (PKG)/mPTP signaling pathway that can increase the resistance of cardiomyocytes to ischemic damage. Materials and methods: Isolated hearts from Wistar rats were perfused with NO donor NOC-18 prior to induction of stop–flow ischemia. To quantify and characterize the phosphoproteins, mitochondrial proteins were resolved and analyzed by two-dimensional gel electrophoresis followed by Pro-Q Diamond phosphoprotein gel staining, excision, trypsin digestions, and mass spectrometry. Quantitative proteomic analysis coupled with liquid chromatography–tandem mass spectrometry was also performed. Results: Mitochondrial protein phosphorylation patterns in NOC-18-pretreated ischemic hearts versus ischemic hearts were compared. Pretreatment of hearts with NOC-18 caused changes in mitochondrial phosphoproteome after ischemia which involved modifications of 10 mitochondrial membrane-bound and 10 matrix proteins. Among them, α-subunit of ATP synthase and adenine nucleotide (ADP/ATP) translocase 1, both of which are considered as potential structural components of mPTP, were identified. We also found that treatment of isolated non-ischemic mitochondria with recombinant PKG did not cause the same protein phosphorylation as pretreatment of hearts with NOC-18. Conclusions: Our study suggests that pretreatment of hearts with NOC-18 causes changes in mitochondrial phosphoproteome after ischemia which involves modifications of certain proteins thought to be involved in the regulation of mPTP opening and intracellular redox state. These proteins may be potential targets for pharmacological preconditioning of the heart.

Quantitative monitoring of His and Asp phosphorylation in a bacterial signaling system by using Phos-tag Magenta/Cyan fluorescent dyes.

In the bacterial signaling mechanisms known as two-component systems (TCSs), signals are generally conveyed by means of a His-Asp phosphorelay. Each system consists of a histidine kinase (HK) and its cognate response regulator. Because of the labile nature of phosphorylated His and Asp residues, few approaches are available that permit a quantitative analysis of their phosphorylation status. Here, we show that the Phos-tag dye technology is suitable for the fluorescent detection of His- and Asp-phosphorylated proteins separated by SDS-PAGE. The dynamics of the His-Asp phosphorelay of recombinant EnvZ-OmpR, a TCS derived from Escherichia coli, were examined by SDS-PAGE followed by simple rapid staining with Phos-tag Magenta fluorescent dye. The technique permitted not only the quantitative monitoring of the autophosphorylation reactions of EnvZ and OmpR in the presence of adenosine triphosphate (ATP) or acetyl phosphate, respectively, but also that of the phosphotransfer reaction from EnvZ to OmpR, which occurs within 1min in the presence of ATP. Furthermore, we demonstrate profiling of waldiomycin, an HK inhibitor, by using the Phos-tag Cyan gel staining. We believe that the Phos-tag dye technology provides a simple and convenient fluorometric approach for screening of HK inhibitors that have potential as new antimicrobial agents.

Detecting miRNAs Using DNA Nanoswitches

miRNAs are short, noncoding RNAs (18–25 nt) and their expression levels in tissues, cells, and bodily fluids serve as biomarkers for diagnosing cellular events or diseases. The miRNA-activated conditional looping of engineered switches (miRacles) assay is based on a conformational change of a DNA nanoswitch. Recognition and binding of a target miRNA to the detectors reconfigures the switch from the linear ‘off’ state to the looped ‘on’ state. The two states can be quantified using standard agarose gel electrophoresis and gel stains, where the detection signal arises from the intensity of the looped nanoswitch.

Vitellogenin in the European cave salamander, Proteus anguinus: Its characterization and dynamics in a captive female as a basis for non-destructive sex identification.

Vitellogenin (Vtg) is a precursor protein of egg yolk proteins in oviparous and ovoviviparous vertebrates. Except in a case of exposure to estrogenic endocrine disruptors, Vtg is a female-specific protein and could be used as a molecular marker for sex identification. This would be especially useful in the case of the endangered European cave salamander Proteus anguinus in which sexes are indistinguishable according to external morphology, which hinders the establishment of a successful captive breeding program. Here we describe the identification, partial characterization, and purification of Vtg from P. anguinus. Vtg was identified in the plasma of a vitellogenic proteus female with visible oocytes. The identification of this protein was accomplished by mass spectrometry analysis. Two-dimensional gel electrophoresis revealed proteus Vtg as a mix of 190 kDa isoforms with isoelectric points in the pH range 5.3-6.0. Vtg was purified from proteus blood by gel filtration followed by anion-exchange chromatography. Using specific staining of SDS-PAGE gels, the Vtg was found to be phosphorylated and lipidated. Unlike the case in some other aquatic vertebrates, in P. anguinus, Vtg was not present in detectable amounts in cutaneous mucus. Degradation of oocytes in the captive vitellogenic female was accompanied by simultaneous decrease of Vtg concentration. Over a period of 10 months, the concentration of Vtg dropped from maximal to sub-detectable. Our results show that Vtg is a promising molecular marker for sex identification and ovary maturation in P. anguinus, which could contribute to the development of a viable program for captive reproduction of this unique species.

Phosphoproteins Involved in the Inhibition of Apoptosis and in Cell Survival in the Leiomyoma.

Uterine leiomyomas are benign smooth muscle cell tumors originating from the myometrium. In this study we focus on leiomyoma and normal myometrium phosphoproteome, to identify differentially phosphorylated proteins involved in tumorigenic signaling pathways, and in anti-apoptotic processes and cell survival. We obtained paired tissue samples of seven leiomyomas and adjacent myometria and analyzed the phosphoproteome by two-dimensional gel electrophoresis (2-DE) combined with immobilized metal affinity chromatography (IMAC) and Pro-Q Diamond phosphoprotein gel stain. We used mass spectrometry for protein identification and Western blotting for 2-DE data validation. Quantities of 33 proteins enriched by the IMAC approach were significantly different in the leiomyoma if compared to the myometrium. Bioinformatic analysis revealed ten tumorigenic signaling pathways and four phosphoproteins involved in both the inhibition of apoptosis and cell survival. Our study highlights the involvement of the phosphoproteome in leiomyoma growth. Further studies are needed to understand the role of phosphorylation in leiomyoma. Our data shed light on mechanisms that still need to be ascertained, but could open the path to a new class of drugs that not only can block the growth, but could also lead to a significant reduction in tumor size.

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The classic silver stains have certain drawbacks, such as high background staining, poor protein recovery, low reproducibility, a narrow linear dynamic range for quantification, and limited compatibility with mass spectrometry (MS). Now, with the use of a fluorogenic Ag+ probe, TPE-4TA, we developed a fluorescent silver staining method for the total protein visualization in polyacrylamide gels. This new stain avoids the troublesome silver reduction step in traditional silver stains. Moreover, the fluorescent silver stain demonstrates good reproducibility, sensitivity, and linear quantification in protein detection, making it a useful and practical protein gel stain.

A program to improve the hand hygiene compliance of Hong Kong preschoolers with an insight into their absenteeism

Hand hygiene prevents cross-infection and is the most effective defense against contagious diseases. Preschoolers, without proper hand hygiene skills, are a vulnerable group to such diseases. The aim of this study was to evaluate whether hand hygiene compliance training of preschoolers in Hong Kong improves their hand hygiene knowledge and performance, and whether the training reduces their absenteeism resulting from influenza symptoms. This was a quasi-experimental study with a pretest-posttest design. A total of 110 preschoolers participated in a program consisting of 4 training sessions on hand hygiene. Before and after the program, their hand hygiene knowledge was assessed by 10 true or false questions, and their handwashing skills were assessed by photos of their hands taken before and after handwashing. Before handwashing, a fluorescent stain gel was used to cover their hands. To determine whether a causal relationship existed between compliance and absenteeism, the preschoolers' absenteeism data were collected during a period of 3 months for analysis. Significant differences were found in hand hygiene knowledge on handwashing steps, duration, and after playing with toys. Percentage increases were recorded for 8 questions. There was significant improvement in hand hygiene performance on the fronts and the backs of fingers, thumbs, and fingertips (P < .05). The findings showed a decrease in absence rates with influenza symptoms for the month before (31%), during (30%), and after (25%) the completion of the program in all participating schools. After the training program, the participants' hand hygiene knowledge and performance considerably improved. They washed their hands more thoroughly and there was a significant reduction in absences owing to influenza. Good cooperation between schools and families is found to be important in helping students build positive attitudes and habits, even in performing simple routines like hand hygiene.

Detection of Posttranslational Modifications by Fluorescent Staining of Two-Dimensional Gels.

Posttranslational modifications (PTMs) are key to the regulation of functional activities of proteins. Quantitative and qualitative information about PTM stages of proteins is crucial for the discovery of disease biomarkers. Fluorescent dyes specifically staining protein PTMs such as phosphorylation and glycosylation enable the specific detection of protein regulations taking place with respect to these modifications. Activity and molecular interactions of many proteins are determined by their extent of phosphorylation. In our search for biomarkers of neurodegenerative diseases such as multiple sclerosis (MS), using an animal model, experimental autoimmune encephalomyelitis (EAE), we have applied the phosphorylation-specific fluorescent dye, ProQ Diamond, to study changes taking place in the phosphoproteome. Subsequent colloidal Coomassie staining of the same gels detects the changes at the whole proteome level. We have detected many changes taking place in the CNS tissue of the EAE animals at the whole proteome as well as at the phosphoproteome level resulting in valuable insights into the pathophysiological mechanism of EAE and MS.

Proteomic identification of an alpha class glutathione S-transferase in freshwater snails (Bulinus globosus)

Abstract Bulinus globosus is a freshwater snail that serves as intermediate host for Schistosoma haematobium and is known to be drought-tolerant. A previous report on the alteration in the status of glutathione S-transferase (GST) in B. globosus during aestivation and recovery, suggested that GST might be involved in the adaptation of the organism to drought. Therefore, the present study aims to characterize GST isoforms that are displayed in snails under drought stress, sampled during the dry season, and those under recovery, sampled during the rainy season. Our data show that the hepatopancreas contained the highest level of GST compared to foot muscle, hemolymph, and visceral mass. GST activity in the hepatopancreas of snails under drought stress was about 2-3 times higher than that of the recovered snails. Activity staining of a polyacrylamide gel revealed that B. globosus both in the active and inactive state has at least three forms of GST. Based on SDS-PAGE, the multiplicity of the main GSTs in the hepatopancreas of B. globosus (BgGSTs) was further revealed by the presence of five protein bands. In addition, LC-MS/MS analysis of BgGST 2, 3 and 4 revealed the expression of alpha GST. In conclusion, B. globosus exhibits tissue-specific expression of multiple GSTs with elevated activities when the organism is under stress. Elevated activities coupled with an alpha GST class expression accentuate the role of BgGSTs as an antioxidant defense system, in particular under stress conditions.

Ursolic Acid Induces Apoptotic Cell Death Through AIF and Endo G Release Through a Mitochondria-dependent Pathway in NCI-H292 Human Lung Cancer Cells In Vitro

Ursolic acid (UA), a triterpene compound present in natural plants, has been shown to induce cytotoxic effects on many human cancer cells through induction of cell-cycle arrest and apoptosis. This study investigated the effects of UA on human lung cancer NCI-H292 cells in vitro. Flow cytometric assay was used to measure the percentage of cell viability, apoptotic cell death by double staining of annexin V and propidium iodide (PI), production of reactive oxygen species (ROS) and Ca2+, and mitochondriaI membrane potential (Ψm). UA-induced chromatin condensation and DNA fragmentation were examined by 4',6-diamidino-2-phenylindole staining and DNA gel electrophoresis, respectively. Western blotting was used to examine the changes of apoptosis-associated protein expression in NCI-H292 cells. UA reduced cell viability and induced apoptotic cell death. UA increased Ca2+ production, reduced Ψm, but did not affect ROS production in NCI-H292 cells. UA increased apoptosis-inducing factor (AIF) and endonuclease G in NCI-H292 cells. Based on these observations, we suggest UA induces apoptotic cell death via AIF and Endo G release through a mitochondria-dependent pathway in NCI-H292 cells.

Synthesis and Biological Uses of A3B Type Water‐Soluble Phthalocyanine Alternate to Alcian Blue

AbstractThis study describes the synthesis and characterization of new non‐symmetric cationic phthalocyanine derivatives and their capability for use in staining of microscopic normal and pathological tissue sections, polyacrylamide gel staining, and spectrophotometric analyses. These new synthesized compounds have specifically stained the glycosaminoglycans (GAGs) in the matrix of cartilage tissue of the trachea. In addition, the mucus produced by the stomach, the small intestine and the colon tissue were stained with non‐symmetrical cationic phthalocyanines comparable to the Alcian Blue 8GX. In addition, the determination of GAGs and glycoproteins in polyacrylamide gels has also been evaluated. Besides, the usage of these compounds to spectrophotometrically measure GAGs in biological fluids was also investigated. We have found that 6 and 7 are good alternatives to Alcian Blue 8GX due to comparable staining performance and interaction with biological molecules, especially acidic components such as acidic GAGs and mucin. These dyes have good solubility with quaternary ammonium groups effecting their binding performance to biological molecules.

Identification of phosphorylation proteins in response to water deficit during wheat flag leaf and grain development

BackgroundWheat (Triticum aestivum L.) serves as important grain crop widely cultivated in the world, which is often suffered by drought stress in natural conditions. As one of the most important post translation modifications, protein phosphorylation widely participates in plant abiotic stress regulation. In this study, we performed the first comparative analysis of phosphorylated protein characterization in flag leaves and developing grains of elite Chinese bread wheat cultivar Zhongmai 175 under water deficit by combining with proteomic approach and Pro-Q Diamond gel staining.ResultsField experiment showed that water deficit caused significant reduction of plant height, tiller number, ear length and grain yield. 2-DE and Pro-Q Diamond gel staining analysis showed that 58 proteins were phosphorylated among 112 differentially accumulated proteins in response to water deficit, including 20 in the flag leaves and 38 in the developing grains. The phosphorylated proteins from flag leaves mainly involved in photosynthesis, carbohydrate and energy metabolism, while those from developing grains were closely related with detoxification and defense, protein, carbohydrate and energy metabolism. Particularly, water deficit resulted in significant downregulation of phosphorylated modification level in the flag leaves, which could affect photosynthesis and grain yield. However, some important phosphorylated proteins involved in stress defense, energy metabolism and starch biosynthesis were upregulated under water deficit, which could benefit drought tolerance, accelerate grain filling and shorten grain developing time.ConclusionsThe modification level of those identified proteins from flag leaves and grains had great changes when wheat was suffered from water deficit, indicating that phosphoproteins played a key role in response to drought stress. Our results provide new insights into the molecular mechanisms how phosphoproteins respond to drought stress and thus reduce production.

Cationic Electrophoresis.

Denaturing, discontinuous electrophoresis in the presence of SDS has become a standard method for the protein scientist. However, there are situations where this method produces suboptimal results. In these cases electrophoresis in the presence of positively charged detergents like cetyltrimethylammonium bromide (CTAB) may work considerably better. Methods for electrophoresis and staining of such gels are presented.

A tetrad apparatus for protein gel casting, electrophoresis, staining, and scanning techniques with dual sensors for automatic detection of gel polymerization and protein migration.

Recent advancements in biochemical sciences have facilitated researchers to explore the structure and function of macro molecules in a cell. PAGE is one of the most favored and adapted laboratory techniques. Due to its simple and economical procedures, several variants or new modifications are routinely observed in the basic electrophoresis technique that comprises gel casting, electrophoresis, staining, and imaging process which consequently necessitates additional apparatuses/components in the laboratory. Operation of these additional apparatuses/components lengthens the pre- and postelectrophoresis procedures involving many intermittent tedious and time-consuming steps. A universal apparatus that can facilitate all such associated techniques is lacking and is of utmost importance for fast and effective results. An apparatus that can perform synchronized action of slab gel casting (16×16cm), electrophoresis (SDS-PAGE), dye staining (Coomassie), and imaging (scanning) techniques with real-time monitoring through sensor technology is described in this article. The estimated cost (∼$150) of fabrication of the apparatus is very economical and simple assembly procedure of the main apparatus can be completed within ∼30min after fabrication.

Real-Time PCR for Ceratocystis platani detection: in-depth validation to assess the diagnostic potential and include additional technical options

A high-performing detection method is essential to safeguard those countries that are still unaffected by canker stain, a devastating disease of Platanus spp. caused by Ceratocystis platani. We previously developed EvaGreen and Taqman-based Real-Time PCR to detect this pathogen, but in-depth validation is needed to guarantee users about its effectiveness and promote its utilization. In this work we present a validation study designed according to EPPO standards, focusing on the analytical and diagnostic sensitivity and specificity. We extend its technical application using SYBR Green. By performing standard curves and eight-replication-based experiments, we established the detection limit at 3 fg C. platani gDNA per PCR reaction. The repeatability and the operator-based reproducibility of the Real-Time PCR was demonstrated. Different gDNA extraction events by different operators and different gDNA extraction modalities did not affect the detection limit. The detection limit threshold cycle was earliest with SYBR Green, followed by Taqman, and EvaGreen. Spiking 6 µl DNA extractions of uninfected, necrotized wood with 3 fg C. platani gDNA confirmed the detection limit: 3 fg C. platani gDNA per PCR reaction, i.e., 0.5 fg gDNA per µl of wood extract. The assays tolerated 6 µl of necrotic C. platani-infected wood extracts without inhibition except for long-dead wood samples, while the 2 µl dose consistently allowed for successful detection. Detection of the pathogen in infected samples showed the highest diagnostic sensitivity with the SYBR Green assay. Agarose gel electrophoresis and staining was validated for visualizing amplicons, even at the detection limit. The specificity of the method was tested against 23 isolates representing the diversity of Ceratocystidaceae, and most species were not detected at 5 ng gDNA. However, some South American strains of the C. fimbriata complex were detected at doses as low as 5 fg. The method remains specific for C. platani detection as no other Ceratocystidaceae are known to colonize plane tree and the species within the geographic range of canker stain of plane tree were only detected at 500 pg or more gDNA. This work paves the way for a performance study of inter-laboratory comparisons.

Real-Time Visualization of in Vitro Transcription of a Fluorescent RNA Aptamer: An Experiment for the Upper-Division Undergraduate or First-Year Graduate Laboratory

A teaching laboratory experiment is described where students prepare in vitro transcription reactions of a fluorescent RNA aptamer, named Broccoli, and observe the production of the aptamer in real-time on a fluorescence plate reader. Alternate visualization methods with minimal costs are also described for laboratories lacking this instrumentation. Two optional experiments are also described. Optional Experiment 1 involves purification of RNA transcription reactions using a commercial spin column kit and having students correlate cleanup kit yield with transcribed aptamer fluorescence. Optional Experiment 2 involves running a polyacrylamide gel of the transcription reaction with a ladder, followed by staining with (Z)-4-(3′,5′-difluoro-4′-hydroxybenzylidene)-2-methyl-1-(2″,2″,2″-trifluoroethyl)-1H-imidazol-5-(4H)-one (DFHBI-1T) (selective for Broccoli) and a second stain with SYBR Gold (nonselective, allowing for simultaneous visualization of Broccoli and ladder). This experiment has the practical advantage of enabling aptamer visualization in laboratories without a fluorescence spectrometer or plate reader, as well as the pedagogical benefit of demonstrating specific activation of the fluorescence of a small molecule by an RNA aptamer in another context (gel staining). Each experiment allows students to perform straightforward, easily understood teaching laboratory experiments, including key concepts in cellular imaging, and RNA biochemistry widely employed in biochemical research.