Name: a colorimetric assay of citrate synthase activity in drosophila melanogaster
Uploaded: 2020-01-16T12:00:00
Description: Watch this Scientific Journal Video about A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster at JoVE.com

This work was supported by the grants from the National Natural Science Foundation of China (31401013 and 31471010), the Science and Technology Commission of Shanghai Municipality, Shanghai Pujiang Program (14PJ1405900), and Natural Science Foundation of Shanghai (19ZR1446400).

1. Colorimetric Citrate Synthase Activity Assay for D. melanogaster16
<ol>
	<li>Collect ten adult flies for each sample. Collect at least triplicate samples for each genotype.</li>
	<li>Prepare 500 &#181;L of ice-cold extraction buffer containing 20 mM HEPES (pH = 7.2), 1 mM EDTA, and 0.1% triton X-100 in a 1.5 mL test tube for each sample.</li>
	<li>Anesthetize adult flies with CO2 on an anesthesia pad and isolate the desired tissues. To isolate the adult fly thoraxes, for e...

<ol>
	<li>Yee, C., Yang, W., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+intrinsic+apoptosis+pathway+mediates+the+pro-longevity+response+to+mitochondrial+ROS+in+C.+elegans.">The intrinsic apoptosis pathway mediates the pro-longevity response to mitochondrial ROS in C. elegans.</a> Cell. 157 (4), 897-909 (2014).</li><li>Sarasija, S., Norman, K. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+gamma-Secretase+Independent+Role+for+Presenilin+in+Calcium+Homeostasis+Impacts+Mitochondrial+Function+and+Morphology+in+Caenorhabditis+elegans.">A gamma-Secretase Independent Role for Presenilin in Calcium Homeostasis Impacts Mitochondrial Function and Morphology in Caenorhabditis elegans.</a> Genetics. 201 (4), 1453-1466 (2015).</li><li>Oxenoid, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Architecture+of+the+mitochondrial+calcium+uniporter.">Architecture of the mitochondrial calcium uniporter.</a> Nature. 533 (7602), 269-273 (2016).</li><li>Hekimi, S., Wang, Y., Noe, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+ROS+and+the+Effectors+of+the+Intrinsic+Apoptotic+Pathway+in+Aging+Cells:+The+Discerning+Killers!.">Mitochondrial ROS and the Effectors of the Intrinsic Apoptotic Pathway in Aging Cells: The Discerning Killers!.</a> Frontiers in Genetics. 7, 161 (2016).</li><li>Kim, H. E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lipid+Biosynthesis+Coordinates+a+Mitochondrial-to-Cytosolic+Stress+Response.">Lipid Biosynthesis Coordinates a Mitochondrial-to-Cytosolic Stress Response.</a> Cell. 166 (6), 1539-1552 (2016).</li><li>Wang, Y., Hekimi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction+and+longevity+in+animals:+Untangling+the+knot.">Mitochondrial dysfunction and longevity in animals: Untangling the knot.</a> Science. 350 (6265), 1204-1207 (2015).</li><li>Ray, A., Martinez, B. A., Berkowitz, L. A., Caldwell, G. A., Caldwell, K. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mitochondrial+dysfunction,+oxidative+stress,+and+neurodegeneration+elicited+by+a+bacterial+metabolite+in+a+C.+elegans+Parkinson's+model.">Mitochondrial dysfunction, oxidative stress, and neurodegeneration elicited by a bacterial metabolite in a C. elegans Parkinson's model.</a> Cell Death &amp; Disease. 5, e984 (2014).</li><li>Tronstad, K. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regulation+and+quantification+of+cellular+mitochondrial+morphology+and+content.">Regulation and quantification of cellular mitochondrial morphology and content.</a> Current Pharmaceutical Design. 20 (35), 5634-5652 (2014).</li><li>Wiegand, G., Remington, S. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Citrate+synthase:+structure,+control,+and+mechanism.">Citrate synthase: structure, control, and mechanism.</a> Annual Review of Biophysics and Biophysical Chemistry. 15, 97-117 (1986).</li><li>Lopez-Lluch, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Calorie+restriction+induces+mitochondrial+biogenesis+and+bioenergetic+efficiency.">Calorie restriction induces mitochondrial biogenesis and bioenergetic efficiency.</a> Proceedings of the National Academy of Sciences of the United States of America. 103 (6), 1768-1773 (2006).</li><li>MacInnis, M. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Superior+mitochondrial+adaptations+in+human+skeletal+muscle+after+interval+compared+to+continuous+single-leg+cycling+matched+for+total+work.">Superior mitochondrial adaptations in human skeletal muscle after interval compared to continuous single-leg cycling matched for total work.</a> Journal of Physiology. 595 (9), 2955-2968 (2017).</li><li>Gillen, J. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Twelve+Weeks+of+Sprint+Interval+Training+Improves+Indices+of+Cardiometabolic+Health+Similar+to+Traditional+Endurance+Training+despite+a+Five-Fold+Lower+Exercise+Volume+and+Time+Commitment.">Twelve Weeks of Sprint Interval Training Improves Indices of Cardiometabolic Health Similar to Traditional Endurance Training despite a Five-Fold Lower Exercise Volume and Time Commitment.</a> PLoS One. 11 (4), e0154075 (2016).</li><li>Mukherjee, S., Basar, M. A., Davis, C., Duttaroy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Emerging+functional+similarities+and+divergences+between+Drosophila+Spargel/dPGC-1+and+mammalian+PGC-1+protein.">Emerging functional similarities and divergences between Drosophila Spargel/dPGC-1 and mammalian PGC-1 protein.</a> Frontiers in Genetics. 5, 216 (2014).</li><li>Mukherjee, S., Duttaroy, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Spargel/dPGC-1+is+a+new+downstream+effector+in+the+insulin-TOR+signaling+pathway+in+Drosophila.">Spargel/dPGC-1 is a new downstream effector in the insulin-TOR signaling pathway in Drosophila.</a> Genetics. 195 (2), 433-441 (2013).</li><li>Diop, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PGC-1/Spargel+Counteracts+High-Fat-Diet-Induced+Obesity+and+Cardiac+Lipotoxicity+Downstream+of+TOR+and+Brummer+ATGL+Lipase.">PGC-1/Spargel Counteracts High-Fat-Diet-Induced Obesity and Cardiac Lipotoxicity Downstream of TOR and Brummer ATGL Lipase.</a> Cell Reports. 10 (9), 1572-1584 (2015).</li><li>Rera, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modulation+of+longevity+and+tissue+homeostasis+by+the+Drosophila+PGC-1+homolog.">Modulation of longevity and tissue homeostasis by the Drosophila PGC-1 homolog.</a> Cell Metabolism. 14 (5), 623-634 (2011).</li><li>Wei, P., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=RNF34+modulates+the+mitochondrial+biogenesis+and+exercise+capacity+in+muscle+and+lipid+metabolism+through+ubiquitination+of+PGC-1+in+Drosophila.">RNF34 modulates the mitochondrial biogenesis and exercise capacity in muscle and lipid metabolism through ubiquitination of PGC-1 in Drosophila.</a> Acta Biochimica et Biophysica Sinica (Shanghai). 50 (10), 1038-1046 (2018).</li><li>Tennessen, J. M., Barry, W. E., Cox, J., Thummel, C. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Methods+for+studying+metabolism+in+Drosophila.">Methods for studying metabolism in Drosophila.</a> Methods. 68 (1), 105-115 (2014).</li><li>Bosco, G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+oxygen+concentration+and+pressure+on+Drosophila+melanogaster:+oxidative+stress,+mitochondrial+activity,+and+survivorship.">Effects of oxygen concentration and pressure on Drosophila melanogaster: oxidative stress, mitochondrial activity, and survivorship.</a> Archives of Insect Biochemistry and Physiology. 88 (4), 222-234 (2015).</li></ol>

Metabolic studies using Drosophila as a model must take into consideration the genetic background, diet, and stock maintenance of the flies18. To avoid the effects of different genetic backgrounds on the measurement of citrate synthase activity, different strains of Drosophila should be backcrossed to the control strain for 10 generations. The genetic background of all Drosophila strains used in our experiments is w1118, so we used w1118</sup...

Mitochondria are best known as the power-producing organelles in most eukaryotic organisms, which produce the energy currency, ATP, through the tricarboxylic acid cycle (i.e., Krebs cycle) and oxidative phosphorylation. Mitochondria are also found to play important roles in a lot of other physiological processes, such as regulation of apoptosis1, Ca2+ homeostasis2,3, reactive oxidation species (ROS) generation4, and endoplasmic reticulum (ER)-stress response5. Mitochondrial dysfunction can affect any organ in the body at any age and is a primary cause of metabolic, aging-related6, and neurodegenerative diseases7. Intact mitochondria are mechanistically related to mitochondrial function. Thus, proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function8. Citrate synthase, a rate-limiting enzyme in the first step of the tricarboxylic acid cycle9, is localized in the mitochondrial matrix within eukaryotic cells, and thus can be used as a quantitative marker for the presence of intact mitochondrial mass9,10. Citrate synthase activity can also be used as a normalization factor for intact mitochondrial proteins11,12.
The fruit fly, Drosophila melanogaster, is an excellent model system for studying mitochondrial function, as many molecules and pathways that play pivotal roles in mitochondria are evolutionarily conserved from Drosophila to humans13,14,15. Here, we present a protocol for a fast and simple method for measurement of citrate synthase activity by a colorimetric assay in Drosophila tissue homogenates16 in a 96 well plate format. In the citrate synthase activity assay, citrate synthase in Drosophila tissue homogenate catalyzes the reaction of oxaloacetate with acetyl coenzyme A (acetyl CoA) to form the citrate CoA-SH and H+. CoA-SH subsequently reacts with 5,5&#39;-dithiobis-(2-nitrobenzoic acid) (DTNB) to generate a colored product, 2-nitro-5-thiobenzoate (TNB), which can be easily measured spectrophotometrically at 412 nm. Citrate synthase activity can be reflected by the rate of color production.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfonic acid (HEPES)</td>
			<td>Sigma-Aldrich</td>
			<td>V900477</td>
			<td></td>
		</tr>
		<tr>
			<td>2-Amino-2-(hydroxymethyl)-1,3-propanediol (TRIZMA Base)</td>
			<td>Sigma-Aldrich</td>
			<td>V900483</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetyl-CoA</td>
			<td>Sigma-Aldrich</td>
			<td>A2181</td>
			<td></td>
		</tr>
		<tr>
			<td>Dithio-bis-nitrobenzoic acid (DTNB)</td>
			<td>Sigma-Aldrich</td>
			<td>D8130</td>
			<td></td>
		</tr>
		<tr>
			<td>Ethylenediaminetetraacetic acid (EDTA)</td>
			<td>Sigma-Aldrich</td>
			<td>V900106</td>
			<td></td>
		</tr>
		<tr>
			<td>Oxaloacetate</td>
			<td>Sigma-Aldrich</td>
			<td>O4126</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle</td>
			<td>Sangon</td>
			<td>F619072</td>
			<td></td>
		</tr>
		<tr>
			<td>Pellet pestle motor</td>
			<td>Tiangen</td>
			<td>OSE-Y10</td>
			<td></td>
		</tr>
		<tr>
			<td>Plate reader</td>
			<td>BioTek</td>
			<td>Eon</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein BCA Assay kit</td>
			<td>Beyotime</td>
			<td>P0010S</td>
			<td></td>
		</tr>
		<tr>
			<td>Scissors</td>
			<td>WPI</td>
			<td>14124</td>
			<td></td>
		</tr>
		<tr>
			<td>Triton X-100</td>
			<td>Sangon</td>
			<td>A110694-0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

a colorimetric assay of citrate synthase activity in drosophila melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of physiological processes. Mitochondrial dysfunction is a primary cause of a number of metabolic and neurodegenerative diseases. Intact mitochondria are critical for their proper functioning. The enzyme citrate synthase is localized in the mitochondrial matrix and thus can be used as a quantitative enzyme marker of intact mitochondrial mass. Given that many molecules and pathways that have important functions in mitochondria are highly conserved between humans and Drosophila, and that an array of powerful genetic tools are available in Drosophila, Drosophila serves as a good model system for studying mitochondrial function. Here, we present a protocol for fast and simple measurement of citrate synthase activity in tissue homogenate from adult flies without isolating mitochondria. This protocol is also suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues.

Figure 1 presents an example of the kinetic curves for the OD absorbance at 412 nm over time obtained using the citrate synthase activity colorimetric assay to measure the Drosophila thorax tissue homogenates of different genotypes. It is well known that PGC-1&#945; is a master regulator of mitochondrial biogenesis. PGC-1&#945; is functionally conserved between Drosophila and humans. Drosophila RNF34 (dRNF34) is an E3 ubiquitin liga...

Watch this Scientific Journal Video about A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster at JoVE.com

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

We present a protocol for a colorimetric assay of citrate synthase activity for quantification of intact mitochondrial mass in Drosophila tissue homogenates.

A Colorimetric Assay of Citrate Synthase Activity in Drosophila Melanogaster

Mitochondria play the most prominent roles in cellular metabolism by producing ATP through oxidative phosphorylation and regulating a variety of ...

Drosophila serves as good model system for studying mitochondrial function. Here we present a protocol for measuring citrate synthase activity in tissue homogenate from adult flies. This protocol does not require isolation of mitochondria.It is fast and simple and it is suitable for measuring citrate synthase activity in larvae, cultured cells, and mammalian tissues. Begin by collecting adult flies for each sample, making sure to collect at least triplicate samples for each genotype. Prepare 500 microliters of ice-cold extraction buffer with 20 millimolar HEPES, one millimolar EDTA, and 0.1%triton X-100 in a 1.5-milliliter test tube for each sample.Anesthetize the adult flies with carbon dioxide on an anesthesia pad. To isolate the thoraxes, fix them with a pair of forceps and isolate the fly abdomens by cutting along the border of the thorax and abdomen with a pair of scissors. Collect the fly thoraxes for the citrate synthase activity assay.Transfer 10 adult fly thoraxes to 100 microliters of ice-cold extraction buffer immediately and homogenize them with a pestle on ice. Keep the samples cold by homogenizing for five to 10 seconds on ice, letting them sit on ice for five seconds, then repeating until all tissues are completely homogenized. Aliquot 10 microliters of each homogenized sample into a new tube for protein content measurement, keeping these tubes on ice as well.Add 400 microliters of ice-cold extraction buffer to each remaining sample for a total volume of 500 microliters and pipette up and down gently to mix. For each reaction, add one microliter of diluted cell lysate to 150 microliters of the freshly prepared reaction solution. Mix thoroughly by gently pipetting, making sure to avoid bubble formation, then use a plate reader to measure the absorbance at 412 nanometers every 10 to 30 seconds for four minutes at 25 degrees Celsius.Plot the data as absorbance over time, then calculate the slope for the linear portion of the curve. Finally, divide the reaction rate or slope by the sample protein concentration to normalize the citrate synthase activity. It was hypothesized that knockdown of dRNF34 in Drosophila muscle would increase mitochondrial citrate synthase activity while knockdown of dPGC-1 would reverse this increase.An initial linear enzymatic rate was established for each assay;the slopes of the trendlines represent the maximal reaction rates which are equivalent to the maximal citrate synthase activities of the different genotypes. The maximal citrate synthase activities were calculated from the kinetic curves and normalized by protein concentration. The results demonstrated that the synthase activity of fly thoraxes indeed increased with muscle-specific dRNF34 knockdown and the increase was reversed by muscle-specific dPGC-1 knockdown.When homogenizing the sample, it is important to tip the tube and check the homogenate to make sure that there are no clots and that the tissues in the tube are completely homogenized. After sample collection, all sample treatments should be performed on ice. The citrate synthase activity assay is used to quantify intact mitochondrial mass.Proper quantification of intact mitochondrial mass is very important for evaluating mitochondrial function.

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This protocol describes in vitro and in vivo methods to characterize the chaperone activity of E. coli HdeB, an acid-activated chaperone. Light scattering measurements were used as a convenient read-out for HdeB&#39;s capacity to prevent acid-induced aggregation of an established model client protein, MDH, in vitro. Analytical ultracentrifugation experiments were applied to reveal complex formation between HdeB and its client protein LDH, to shed light into the fate of client proteins upon their return to non-stress conditions. Enzymatic activity assays of the client proteins were conducted to monitor the effects of HdeB on pH-induced client inactivation and reactivation. Finally, survival studies were used to monitor the influence of HdeB&#39;s chaperone function in vivo.

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Bacteria are frequently exposed to environmental changes, such as alterations in pH, temperature, redox status, light exposure or mechanical force. Many ...

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Enveloped viruses such as coronaviruses or influenza virus require proteolytic cleavage of their fusion protein to be able to infect the host cell. Often ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...

Continuous Fluorescence-Based Endonuclease-Coupled DNA Methylation Assay to Screen for DNA Methyltransferase Inhibitors

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) establish and maintain the DNA methylation pattern. Changes to the normal DNA methylation pattern are linked to cancer development and progression, making DNMTs potential cancer drug targets. Thus, identifying and characterizing novel small molecule inhibitors of these enzymes is of great importance. This paper presents a protocol that can be used to screen for DNA methyltransferase inhibitors. The continuous coupled kinetics assay allows for initial velocities of DNA methylation to be determined in the presence and absence of potential small molecule inhibitors. The assay uses the methyl-sensitive endonuclease Gla I to couple methylation of a hemimethylated DNA substrate to fluorescence generation.
This continuous assay allows for enzyme activity to be monitored in real time. Conducting the assay in small volumes in microtiter plates reduces the cost of reagents. Using this assay, a small example screen was conducted for inhibitors of DNMT1, the most abundant DNMT isozyme in humans. The highly substituted anthraquinone natural product, laccaic acid A, is a potent, DNA-competitive inhibitor of DNMT1. Here, we examine three potential small molecule inhibitors &#8212; anthraquinones or anthraquinone-like molecules with one to three substituents &#8212; at two concentrations to describe the assay protocol. Initial velocities are used to calculate the percent activity observed in the presence of each molecule. One of three compounds examined exhibits concentration-dependent inhibition of DNMT1 activity, indicating that it is a potential inhibitor of DNMT1.

DNA methyltransferases are potential cancer drug targets. Here, a protocol is presented to assess small molecules for DNA methyltransferase inhibition. This assay utilizes an endonuclease to couple DNA methylation to fluorescence generation and allows for enzyme activity to be monitored in real time.&#160;

DNA methylation, a form of epigenetic gene regulation, is important for normal cellular function. In cells, proteins called DNA methyltransferases (DNMTs) ...

Measuring Caspase Activity Using a Fluorometric Assay or Flow Cytometry

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators and executioners of apoptosis, the most studied form of programmed cell death. Apoptosis occurs during developmental processes and is a necessary event in tissue homeostasis. Pyroptosis is another form of cell death that utilizes caspases and is a critical process in activating the immune system through the activation of the inflammasome, which results in the release of members of the interleukin-1 (IL-1) family. To assess caspase activity, target substrates can be assessed. However, sensitivity can be an issue when examining single cells or low-level activity. We demonstrate how a fluorogenic substrate can be used with a population-based assay or single-cell assay by flow cytometry. With proper controls, different amino acid sequences can be used to identify which caspases are active. Using these assays, the simultaneous loss of the inhibitors of apoptosis proteins upon tumor necrosis factor (TNF) stimulation has been identified, which primarily induces apoptosis in macrophages rather than other forms of cell death.

The present protocol describes two methods to measure caspase activity through a fluorogenic substrate using flow cytometry or a spectrofluorometer.

The activation of cysteine proteases, known as caspases, remains an important process in multiple forms of cell death. Caspases are critical initiators ...

Smartphone as an Analytical Device to Quantify Protein Using Bradford Assay

RGBradford: Protein Quantitation with a Smartphone Camera

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. Because of its widespread, the limitations and advantages of the Bradford assay have been exhaustively reported, including several modifications of the original method to improve its performance. One of the alterations of the original method is the use of a smartphone camera as an analytical instrument. Taking advantage of the three forms of the Coomassie Brilliant Blue dye that exist in the conditions of the Bradford assay, this paper describes how to accurately quantify protein in samples using color data extracted from a single picture of a microplate. After performing the assay in a microplate, a picture is taken using a smartphone camera, and RGB color data is extracted from the picture using a free and open-source image analysis software application. Then, the ratio of blue to green intensity (in the RGB scale) of samples with unknown concentrations of protein is used to calculate the protein content based on a standard curve. No significant difference is observed between values calculated using RGB color data and those calculated using conventional absorbance data.

Author Spotlight: An Alternative Approach to Protein Quantification by Bradford Assay Using a Smartphone 

This paper provides a protocol for protein quantification using the Bradford assay and a smartphone as an analytical device. Protein levels in samples can be quantified using color data extracted from a picture of a microplate taken with a smartphone.

Protein quantitation is an essential procedure in life sciences research. Amongst several other methods, the Bradford assay is one of the most used. ...