Name: A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources (Video) | JoVE Journal
Uploaded: 2026-01-27T14:00:00.000+00:00
Duration: 10 min 24 s
Description: Learn a fluorescence-based protocol for screening protein synthesis inhibitors from natural sources. This method supports drug discovery research and provides hands-on training for students in medicinal chemistry.

ai_agent_IP

ai_agent

maintenance

instantTrialDurationMinutes

isBiopharma

enableNewMyJoveSectionDesign

educationFeatures

seoMetaApi

ai_copilot_institution_blacklist

ai_chat_institution_blacklist

freeAccessWindow

ssoRoleCapture

scheduledMaintenanceBanner

joveLabsFeatures

disable_autoplay_for_bots

extended_trial_duration

5 minutes

This research was supported by NIGMS grant number R15GM148983, the Louisiana Board of Regents LEQSF(2021-25)-RD-A-05, and part from the U.S. National Science Foundation under grant number OIA-1946231 to F.R.

1. Reagent preparation from kit
NOTE: Adequately mix, spin down, and keep all reagents on ice until use.
<ol>
	<li>Organize and obtain the protein synthesis inhibition kit, PBS buffer, cell culture medium, cell culture treated plates, 15 mL conical sterile polypropylene centrifuge tubes, 2 mL centrifuge tubes, p10, p20, p200, and p1000 pipettes, and appropriate tips.</li>
	<li>Prepare wash buffer.
	<ol>
		<li>Thaw the 10x wash buffer at 37 &#176;C by cupping the vial in the hand. Use the body heat in the hands to thaw and frequently check if thawing is complete.</li>
		<li>Dilute the 10x wash buffer stocks 1:10 in deionized water in a centrifuge tube. Mix by inversion or resuspension by pipetting up and down. Store on ice until use.</li>
	</ol>
	</li>
	<li>Thaw fixative solution at room temperature. Store on ice until use.</li>
	<li>Prepare the permeabilization buffer.
	<ol>
		<li>Thaw the 10x permeabilization buffer at 37 &#176;C by cupping the vial in the hand. Use the body heat in the hands to thaw and frequently check if thawing is complete.</li>
		<li>Dilute the 10x stock 1:10 in deionized water in a centrifuge tube. Mix the components by inversion or resuspension by pipetting up and down. Store on ice until use.</li>
	</ol>
	</li>
	<li>Thaw 400x protein label, 100x fluorescent azide, 100x cycloheximide, DNA stain, and the 20x reducing agent at room temperature. Keep on ice in a dark environment while in use. Cover the dyes with foil or keep them in a 2 &#176;C refrigerator when not in use.</li>
</ol>
2. Labeling controls and experimental cells
NOTE: This protocol was developed using stable adherent, epithelial breast cancer MDA-MB-231 cells. Any adherent cell lines can be used. All steps listed below use a 96-well plate, and all volumes and concentrations are calculated for two test compounds and two controls with a working volume of 100 &#181;L.
<ol>
	<li>Culturing adherent mammalian cells 
	NOTE: Cells should be passed before reaching 80-90% confluency to maintain healthy, exponential growth and prevent contact inhibition or differentiation.</li>
	<li>Thaw a vial of cryopreserved cells to room temperature, then dilute with appropriate medium.</li>
	<li>Centrifuge the suspension to pellet the cells and aspirate the supernatant. Resuspend the pellet in fresh medium and transfer to a new flask at the appropriate cell density.</li>
	<li>Once cells are 80% confluent, remove media and aspirate old growth medium from the flask. Then, rinse cells with sterile PBS to remove residual serum, which inhibits trypsin. Add trypsin-EDTA solution to cover the cell layer and incubate for 3-5 min or until cells detach at 37 &#176;C.</li>
	<li>Add complete medium containing serum to inactivate the trypsin, then transfer the cell suspension to a tube, mix gently, and count the cells.</li>
	<li>Seed cells in screening plates-dilute cells at the appropriate split ratio (e.g., 1:3 or 1:5) into fresh flask(s) with new medium and incubate (typically 37 &#176;C, 5% CO2) to obtain the desired cell concentration. 
	NOTE: For the experiment described here, the cell line was plated at 2 &#215; 104 per well (96-well plate) and incubated at 37 &#176;C for 12 h.</li>
</ol>
3. Addition of compounds
<ol>
	<li>Prepare master mix A.
	<ol>
		<li>Label a centrifuge tube as &#34;A&#34;. Pipette 598.5 &#181;L of culture medium and 1.5 &#181;L of protein label into &#34;A&#34;. Resuspend the mixture to ensure homogeneity by pipetting up and down.</li>
	</ol>
	</li>
	<li>Prepare master mix B.
	<ol>
		<li>Label a centrifuge tube as &#34;B&#34;. Pipette 1.5 &#181;L of protein label, 6.6 &#181;L of cycloheximide, and 591.9 &#181;L of culture medium into &#34;B&#34;. Resuspend the mixture to ensure homogeneity by pipetting up and down.</li>
	</ol>
	</li>
	<li>Treat cells with desired compounds (Figure 1), positive, and negative controls.
	<ol>
		<li>Remove cells from the incubator and remove the growth medium via aspiration. Tilt the plate and aspirate at the side of the well instead of the center to prevent disturbance of the cells.</li>
		<li>Pipette 1 &#181;L of test compound into a well and 1 &#181;L of test compound vehicle (0.1% v/v DMSO) into a well. For duplicate studies, add each sample to a second well and repeat this process. Add 99 &#181;L of master mix A into these wells. Gently pipette at an angle into the side of the well to prevent kinetic disruption of cells. 
		&#8203;NOTE: The maximum DMSO concentration for compound screening depends on the cell type and assay, but general guidelines are: 0.1-0.5% DMSO-generally safe for most cell-based assays with minimal toxicity, and 1% DMSO-commonly used maximum and acceptable for many cell types in short-term assays.</li>
		<li>Pipette 100 &#181;L of master mix B into two wells. Pipette at an angle into the side of the well to prevent disruption of cells.</li>
		<li>Gently tap the plate to achieve through incorporation of the desired components with the cells. Incubate cells at 37 &#176;C for 0.5-2 h.</li>
	</ol>
	</li>
</ol>
4. Termination of the experiment and washing of cells
<ol>
	<li>Remove the culture medium via aspiration. Tilt the plate and aspirate at the side of the well instead of the center to prevent disturbance of the cells.</li>
	<li>Add 100 &#181;L of PBS buffer into each well and centrifuge the plate at 900 &#215; g for 5 min.</li>
	<li>Remove the PBS buffer via aspiration.</li>
</ol>
5. Fixation and permeabilization
<ol>
	<li>Add 100 &#181;L of fixative solution to each well. Incubate the cells at room temperature for 15 min in a dark environment.</li>
	<li>Remove the fixative solution via aspiration. Tilt the plate and aspirate at the side of the well instead of the center.</li>
	<li>Wash cells with 100 &#181;L of wash buffer and remove buffer via aspiration. Repeat this step 2x.</li>
	<li>Add 100 &#181;L of permeabilization buffer to each well and incubate at room temperature for 10 min.</li>
	<li>Remove the permeabilization buffer via aspiration. Tilt the plate and aspirate at the side of the well instead of the center.</li>
</ol>
6. Protein reaction and staining
<ol>
	<li>Prepare the reaction cocktail master mix.
	<ol>
		<li>Add 651 &#181;L of PBS buffer, 7.0 &#181;L of 100x copper reagent, 7 &#181;L of fluorescent azide, and finally, 35 &#181;L of reducing agent to a centrifuge tube. Resuspend this mixture to ensure homogeneity. 
		NOTE: Follow this exact order when adding the reagents. Use within 15 min of preparation.</li>
	</ol>
	</li>
	<li>Add 100 &#181;L of the reaction cocktail to each well. Incubate the cells for 30 min at room temperature in a dark environment to prevent photobleaching.</li>
	<li>Centrifuge cells at 900 &#215; g for 5 min. Balance the centrifuge by adding empty plates to distribute the weight evenly. Remove the mixture via aspiration.</li>
	<li>Wash cells with 100 &#181;L of wash buffer and remove buffer via aspiration. Repeat this step 2x.</li>
</ol>
7. DNA staining
<ol>
	<li>Prepare a 1x dilution of total DNA stain and add 100 &#181;L to each well. Incubate cells at room temperature for 20 min in a dark environment to prevent photobleaching.</li>
	<li>Centrifuge the plate at 900 &#215; g for 5 min. Balance the centrifuge by adding empty plates to distribute the weight evenly. Remove the mixture via aspiration.</li>
	<li>Wash cells with 100 &#181;L of wash buffer and remove buffer via aspiration. Repeat this step 2x.</li>
	<li>Resuspend cells with 100 &#181;L of chilled PBS buffer to prepare them for imaging.</li>
</ol>
8. Imaging
<ol>
	<li>Insert the plate into the plate reader.</li>
	<li>Set magnification to 20x.</li>
	<li>Analyze nucleus stain with the GFP 469/525 nm channel. Use autobinning and autofocus to enhance imaging results.</li>
	<li>Analyze active protein synthesis with the Texas Red 586/647nm channel. Use autobinning and autofocus to enhance imaging results.</li>
</ol>

<ol><li>Merrick, W. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Mechanism+and+regulation+of+eukaryotic+protein+synthesis%5BTitle%5D)+AND+%22Microbiol+Rev%22%5BJournal%5D)">Mechanism and regulation of eukaryotic protein synthesis.</a> Microbiol Rev. 56, 291-315 (1992).</li>
<li>Ng, L., Navarro, A., Law, W. -L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Editorial%3A+evolving+roles+of+piRNAs+in+solid+tumors%5BTitle%5D)+AND+%22Front+Oncol%22%5BJournal%5D)">Editorial: evolving roles of piRNAs in solid tumors.</a> Front Oncol. 13, 1178634(2023).</li>
<li>Brown, J. M., Giaccia, A. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((The+unique+physiology+of+solid+tumors%3A+opportunities+%28and+problems%29+for+cancer+therapy%5BTitle%5D)+AND+%22Cancer+Res%22%5BJournal%5D)">The unique physiology of solid tumors: opportunities (and problems) for cancer therapy.</a> Cancer Res. 58 (7), 1408-1416 (1998).</li>
<li>Kovalski, J. R., Kuzuoglu-Ozturk, D., Ruggero, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Protein+synthesis+control+in+cancer%3A+selectivity+and+therapeutic+targeting%5BTitle%5D)+AND+%22EMBO+J%22%5BJournal%5D)">Protein synthesis control in cancer: selectivity and therapeutic targeting.</a> EMBO J. 41 (8), e109823(2022).</li>
<li>Liu, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Synthetic+silvestrol+analogues+as+potent+and+selective+protein+synthesis+inhibitors%5BTitle%5D)+AND+%22J+Med+Chem%22%5BJournal%5D)">Synthetic silvestrol analogues as potent and selective protein synthesis inhibitors.</a> J Med Chem. 55 (20), 8859-8878 (2012).</li>
<li>Liu, L., Li, Z., Wu, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Harnessing+natural+inhibitors+of+protein+synthesis+for+cancer+therapy%3A+a+comprehensive+review%5BTitle%5D)+AND+%22Pharmacol+Res%22%5BJournal%5D)">Harnessing natural inhibitors of protein synthesis for cancer therapy: a comprehensive review.</a> Pharmacol Res. 209, 107449(2024).</li>
<li>Dias, D. A., Urban, S., Roessner, U. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+historical+overview+of+natural+products+in+drug+discovery%5BTitle%5D)+AND+%22Metabolites%22%5BJournal%5D)">A historical overview of natural products in drug discovery.</a> Metabolites. 2 (2), 303-336 (2012).</li>
<li>Broach, J. R., Thorner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((High-throughput+screening+for+drug+discovery%5BTitle%5D)+AND+%22Nature%22%5BJournal%5D)">High-throughput screening for drug discovery.</a> Nature. 384 (6604 Suppl), 14-16 (1996).</li>
<li>Fang, X., Zheng, Y., Duan, Y., Liu, Y., Zhong, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Recent+advances+in+design+of+fluorescence-based+assays+for+high-throughput+screening%5BTitle%5D)+AND+%22Anal+Chem%22%5BJournal%5D)">Recent advances in design of fluorescence-based assays for high-throughput screening.</a> Anal Chem. 91 (1), 482-504 (2019).</li>
<li>McKay, C. S., Finn, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Click+chemistry+in+complex+mixtures%3A+bioorthogonal+bioconjugation%5BTitle%5D)+AND+%22Chem+Biol%22%5BJournal%5D)">Click chemistry in complex mixtures: bioorthogonal bioconjugation.</a> Chem Biol. 21 (9), 1075-1101 (2014).</li>
<li>Schneider-Poetsch, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Inhibition+of+eukaryotic+translation+elongation+by+cycloheximide+and+lactimidomycin%5BTitle%5D)+AND+%22Nat+Chem+Biol%22%5BJournal%5D)">Inhibition of eukaryotic translation elongation by cycloheximide and lactimidomycin.</a> Nat Chem Biol. 6 (3), 209-217 (2010).</li>
<li>Liu, J., Xu, Y., Stoleru, D., Salic, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Imaging+protein+synthesis+in+cells+and+tissues+with+an+alkyne+analog+of+puromycin%5BTitle%5D)+AND+%22Proc+Natl+Acad+Sci+U+S+A%22%5BJournal%5D)">Imaging protein synthesis in cells and tissues with an alkyne analog of puromycin.</a> Proc Natl Acad Sci U S A. 109 (2), 413-418 (2012).</li>
<li>Liu, K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Evaluating+cell+lines+as+models+for+metastatic+breast+cancer+through+integrative+analysis+of+genomic+data%5BTitle%5D)+AND+%22Nat+Commun%22%5BJournal%5D)">Evaluating cell lines as models for metastatic breast cancer through integrative analysis of genomic data.</a> Nat Commun. 10, 2138(2019).</li>
<li>Ma, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dehydroabietylamine+exerts+antitumor+effects+by+affecting+nucleotide+metabolism+in+gastric+cancer%5BTitle%5D)+AND+%22Carcinogenesis%22%5BJournal%5D)">Dehydroabietylamine exerts antitumor effects by affecting nucleotide metabolism in gastric cancer.</a> Carcinogenesis. 45 (10), 759-772 (2024).</li>
<li>Shinde, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((A+review+of+single-cell+adhesion+force+kinetics+and+applications%5BTitle%5D)+AND+%22Cells%22%5BJournal%5D)">A review of single-cell adhesion force kinetics and applications.</a> Cells. 10 (3), 577(2021).</li>
<li>Vargas-Accarino, E., Herrera-Montávez, C., Ramón y Cajal, s, Aasen, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Spontaneous+cell+detachment+and+reattachment+in+cancer+cell+lines%3A+an+in+vitro+model+of+metastasis+and+malignancy%5BTitle%5D)+AND+%22Int+J+Mol+Sci%22%5BJournal%5D)">Spontaneous cell detachment and reattachment in cancer cell lines: an in vitro model of metastasis and malignancy.</a> Int J Mol Sci. 22 (9), 4929(2021).</li>
<li>Song, L., Hennink, E. J., Young, I. T., Tanke, H. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Photobleaching+kinetics+of+fluorescein+in+quantitative+fluorescence+microscopy%5BTitle%5D)+AND+%22Biophys+J%22%5BJournal%5D)">Photobleaching kinetics of fluorescein in quantitative fluorescence microscopy.</a> Biophys J. 68 (6), 2588-2600 (1995).</li>
<li>Ling, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Dehydroabietylamine+derivatives+target+triple-negative+breast+cancer%5BTitle%5D)+AND+%22Eur+J+Med+Chem%22%5BJournal%5D)">Dehydroabietylamine derivatives target triple-negative breast cancer.</a> Eur J Med Chem. 102, 9-13 (2015).</li>
<li>Gautam, L. N., Ling, T., Lang, W., Rivas, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/pubmed?term=((Anti-proliferative+evaluation+of+monoterpene+derivatives+against+leukemia%5BTitle%5D)+AND+%22Eur+J+Med+Chem%22%5BJournal%5D)">Anti-proliferative evaluation of monoterpene derivatives against leukemia.</a> Eur J Med Chem. 113, 75-80 (2016).</li>
</ol>

The authors have no conflicts of interest to declare.

This protocol provides a fluorometric screening methodology for testing natural products and small molecules to evaluate their ability to inhibit protein synthesis. The simplicity of this protocol enables undergraduate researchers to implement it correctly, allowing them to make substantive contributions to scientific knowledge. The protocol demands a basic comprehension of biological methods and techniques, with steps requiring careful execution to ensure accurate and reliable results.
For in...

Protein synthesis involves highly regulated processes: transcription and translation. During transcription, DNA sequences of genes are copied, producing messenger RNA (mRNA). The enzyme RNA polymerase works to separate the DNA double helix and uses one strand as a guide to build a matching mRNA molecule. Once the mRNA is fully formed, it travels from the nucleus out to the cytoplasm for translation. Translation occurs in the ribosome, wherein mRNA is used as a template to assemble amino acids into a polypeptide chain. This polypeptide chain, upon leaving the exit tunnel of the ribosome, is folded into a functional protein. Transcription is primarily regulated by transcription factors, chromatin modifications, and regulatory RNAs, while translation is regulated by initiation factors, signaling pathways, and RNA stability mechanisms. These regulatory systems allow cells to maintain cellular functions and respond to stress or damage effectively1.
The vast majority of human cancers&#8212;approximately 85%&#8212;manifest as solid tumors2. While these tumors can vary dramatically in their specific features and behaviors, they tend to share certain common physiological traits and patterns, such as upregulated protein synthesis regardless of their etiology or anatomical position3. Cancer cells dysregulate protein synthesis by hijacking translational machinery to support their high energy demands and uncontrolled growth. This reprogramming is a result of dysregulation of nearly all major oncogenic signaling pathways, promoting cell survival, proliferation, and metastasis, while suppressing cell death pathways4. In tumor cells, the machinery for making ribosomes goes into overdrive, enabling increased protein production and rapid cell division. Since this enhanced protein synthesis is crucial for cancer survival, treatments that target and block this process show promise as potential therapies5. Therefore, research focusing on cancer-specific translation factors, unique ribosomal compositions, or altered signaling pathways that regulate protein synthesis could yield more targeted inhibitors with improved therapeutic windows.
Because natural products (NPs) are known for their wide array of biological activity, the development of selective protein synthesis inhibitors for cancer treatment using their scaffolds is a promising approach6,7. NPs can target different components of the protein synthesis machinery, from ribosome binding to interference with initiation factors or elongation processes, making them invaluable starting points in drug development for protein synthesis inhibitors in solid tumors6. The capacity to rapidly screen compounds for desired activity serves as a crucial entry point for drug discovery platforms seeking to efficiently identify NP hits. Typically, high-throughput screening of compounds utilizes fluorescence assays to quickly narrow down lead candidate molecules8.
Scientists worldwide rely on fluorescence-based techniques as essential tools for analyzing biological systems. These approaches are widely used to study cellular processes, allowing researchers to track biomolecular interactions in real time9. When combined with bio-orthogonal click-chemistry reactions, these methods can enable precise visualization of protein synthesis10. The presented fluorescent protein synthesis assay is a non-radioactive technique for assessing protein synthesis that utilizes O-propargyl-puromycin (OPP), a cell-permeable alkyne-containing puromycin analog. Following cellular treatment, OPP becomes incorporated at the C-terminus of elongating polypeptide chains, causing translation termination. These alkyne-labeled truncated proteins are then detected using copper-catalyzed click chemistry coupled with 5 FAM-azide.
This fluorometric assay is a valuable research tool and an accessible chemical biology method for training undergraduate students. By providing an accessible, efficient protocol with minimal data processing, it provides students with hands-on experience in modern drug discovery techniques and accelerates the drug discovery process. As the demand for new protein synthesis inhibitors grows, this approach equips undergraduates with meaningful research opportunities while contributing to advancements in cancer therapeutics.

<table><tbody><tr><td>Agilent Biotek Cytation 1 Cell Imaging Multi-Mode Reader</td><td>Agilent&amp;nbsp;</td><td>BTCYT1V</td><td></td></tr><tr><td>Compound 1 (Dehydroabietylamine, CAS 1446-61-3)</td><td>Enamine</td><td>EN300-7409771</td><td></td></tr><tr><td>Compound 2 (Bakuchiol, CAS 10309-37-2)</td><td>ChemFaces</td><td>CFN99047</td><td></td></tr><tr><td>Heraeus Megafuge 16R</td><td>Thermo Scientific&amp;nbsp;</td><td>75004271</td><td></td></tr><tr><td>Ibidi Gmbh-slide 96-well plate</td><td>Ibidi</td><td>80826</td><td></td></tr><tr><td>MDA-MB-231 Cell Lines</td><td>ATCC</td><td>ATCC HTB-26</td><td></td></tr><tr><td>Phosphate Buffered Saline pH 7.4</td><td>ATCC</td><td>ATCC 30-2200</td><td></td></tr><tr><td>Protein Synthesis Assay Kit (Green)</td><td>Abcam</td><td>Ab239725</td><td></td></tr></tbody></table>

a fluorescence-based protocol for preliminary screening of protein synthesis inhibitors from natural sources

A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources

Natural products represent a rich source of therapeutic compounds with applications across multiple diseases, particularly cancer. By leveraging these biomolecules as structural templates in drug discovery, researchers have achieved both faster development timelines and improved biological effectiveness compared to fully synthetic compounds. The protein synthesis pathway has emerged as a compelling target for natural products, especially given its involvement in hard-to-treat cancers. The potent protein synthesis inhibitor cycloheximide, derived from Streptomyces griseus, exemplifies the potential of natural compounds in this space. As these natural product derivatives are developed into drug-like molecules, confirming their protein synthesis inhibitory activity remains a crucial validation step. The primary aim is to provide an undergraduate-friendly protocol for rapid evaluation of compound inhibition activity. The protocol employs a fluorescent substrate and advanced fluorescence microscopy techniques to systematically detect and measure the inhibitory capabilities of these natural products. The study examined compounds derived from two plant species: Pinus sylvestris and Psoralea corylifolia. Using cycloheximide as a reference inhibitor, both natural products showed varying levels of protein synthesis inhibition.

This assay utilized fluorescence to visualize and quantify different natural products' ability to inhibit protein synthesis. Cycloheximide (CHX) is a known protein synthesis inhibitor whose mode of action involves interfering with the translocation process during elongation11. For this study, living cells were detected with a green, fluorescent nuclear dye. OPP is a derivative of puromycin that covalently binds to nascent proteins12. The MDA-MB-231 cell line served as an ap...

This protocol introduces a fluorometric screening methodology optimized for identifying natural product/small molecule inhibitors of protein synthesis. Its practicality makes it suitable for both undergraduate instruction in drug discovery techniques and implementation in medicinal chemistry campaigns.

Natural products represent a rich source of therapeutic compounds with applications across multiple diseases, particularly cancer. By leveraging these ...

A fluorescence-based protocol for preliminary screening of protein synthesis inhibitors from natural sources. Protein synthesis involves highly regulated processes, transcription and translation. Transcription is primarily regulated by transcription factors, chromatin modifications, and regulatory RNAs.While translation is regulated by initiation factors, signaling pathways and RNA stability mechanisms. These regulatory systems allow cells to maintain cellular functions and respond to stress or damage effectively. Cancer cells dysregulate protein synthesis by hijacking translational machinery to support their high energy demands and uncontrolled growth.This reprogramming is a result of dysregulation of nearly all major oncogenic signaling pathways, promoting cell survival, proliferation and metastasis while suppressing cell death pathways. Since this enhanced protein synthesis is crucial for cancer survival, treatments that target and block this process show promise as potential therapies. To identify protein synthesis inhibitors, the following protein synthesis inhibition fluorescence assay can be utilized.Prior to starting the experiment, in order to obtain reliable results, compounds must be administered at an appropriate concentration beneath its toxicity threshold. To establish a starting concentration, a cytotoxicity assay should be performed to determine the compound's EC 50. If this is not feasible, compounds in this assay can be tested in a dose dependent manner to determine the most effective concentration parameters.If the compounds concentration triggers cell death, testing at reduced concentrations becomes necessary. It is also important to protect dyes from light at all times when not in use. Extended light exposure will result in photobleaching, which leads to compromised data quality.Step 3.1, prepare master mix A, label a centrifuge tube as A then pipette 598.5 microliters of culture medium, and 1.5 microliters of protein label into A.Resuspend this mixture to ensure homogeneity by pipetting up and down. Now prepare master mix B.Label a centrifuge tube as B, then pipette 1.5 microliters of protein label, 6.6 microliters of cycloheximide and 591.9 microliters of culture medium into B.Resuspend this mixture to ensure homogeneity by pipetting up and down. After pipetting one microliter of test compound and one microliter of vehicle into two wells, gently pipette 99 microliters of master mix A into both of these wells, pipette at an angle into the side of the well to prevent kinetic disruption of cells.Pipette 100 microliters of master mix B into two untreated wells. Pipette at an angle into the side of the well to prevent kinetic disruption of cells. Gently tap the plate to achieve thorough incorporation of the desired components within the cells.Incubate cells at 37 degrees Celsius for 30 minutes to two hours. Remove the cell culture medium via aspiration. Tilt the plate and aspirate at the side of the well instead of the center to prevent kinetic disturbance of cells.Then add 100 microliters of PBS buffer into each well and centrifuge the plate at 900g for five minutes. After centrifuging, remember to remove the PBS buffer via aspiration. Step five, fixation and permeabilization.Add 100 microliters of fixative solution to each well. Then incubate the cells at room temperature for 15 minutes in a dark environment. Then aspirate the cells.Wash the cells with 100 microliters of wash buffer and remove via aspiration. Repeat this twice. Add 100 microliters of permeabilization buffer to each well.After this, incubate at room temperature for 10 minutes. Remove the permeabilization buffer via aspiration. Step six, protein reaction and staining.Prepare the reaction cocktail master mix. Add 651 microliters of PBS buffer to a centrifuge tube. Then add seven microliters of 100x copper reagent.Then seven microliters of fluorescent azide. And finally, add 35 microliters of reducing agent to the centrifuge tube. Follow this exact order when adding the reagents, then add 100 microliters of the reaction cocktail to each well.Incubate the cells for 30 minutes in a dark environment at room temperature to prevent photobleaching. Centrifuge the cells at 900g for five minutes. Aspirate, wash the cells and then aspirate again.Prepare a 1x dilution of total DNA stain and add 100 microliters to each well. Incubate cells at room temperature for 20 minutes in a dark environment to prevent photobleaching. Centrifuge the plate at 900g for five minutes.Wash the cells with 100 microliters of wash buffer, remove via aspiration. Repeat this step twice. Finally, resuspend the cells with 100 microliters of chilled PBS buffer to prepare them for imaging.Step eight, imaging. Insert the plate into the plate reader and set magnification to 20 x. Analyze the nucleus stain with the GFP 469/525 nanometer channel.Use auto-bending and auto-focus to enhance imaging results. Analyze active protein synthesis with the Texas Red 586/647 nanometer channel. Use auto-bending and auto-focus to enhance imaging results.Fluorescent probes are sensitive to photobleaching, which is a phenomenon in which a radiation with light causes fluorophores to lose their fluorescent properties. Dyes should be protected from light at all times when not in use. Additionally, the number of scans within each well and time under irradiated light need to be limited.Extended light exposure will result in photobleaching, which ultimately leads to compromised data quality. Let's look at the representative results. Live cells here are seen in green and active protein synthesis is indicated by red.Known protein synthesis inhibitor cycloheximide results in a large decrease in protein synthesis as seen in figure 1B compared to the negative control DMSO in figure 1A, there is less red fluorescent light emitted. Treatment with compound one as seen in figure 1C resulted in either cell detachment or cell death. Thus, we cannot make any definitive claims on its protein synthesis inhibition activity.This warrants further dose response studies to try and determine if it inhibits protein synthesis at lower concentrations. Compound two displays moderate protein synthesis inhibition as indicated in figure 1D. This fluorometric assay is a valuable research tool and an accessible chemical biology method for training undergraduate students.By providing an accessible, efficient protocol with minimal data processing, it provides students with hands-on experience and modern drug discovery techniques and accelerates the drug discovery process. The protocol employs a fluorescent substrate and advanced fluorescent microscopy techniques to systematically detect and measure the inhibitory capabilities of natural product compounds one and two. Using cycloheximide as a reference inhibitor, both natural products showed varying levels of protein synthesis inhibition.As the demand for new protein synthesis inhibitors grows, this approach equips undergraduates with meaningful research opportunities while contributing to advancements in cancer therapeutics.

a-fluorescence-based-protocol-for-preliminary-screening-protein

Research

JoVE Journal

Cancer Research

In this video, we demonstrate the amplified luminescent proximity homogeneous assay—a bead-based proximity assay. It utilizes homogeneously-sized acceptor and donor beads, immobilized with two proteins that tend to interact to screen potential small molecules inhibiting protein interactions.

Amplified Luminescent Proximity Homogeneous Assay: A Bead-Based Proximity Assay to Screen Small Molecules Inhibiting Protein-Protein Interactions

This video demonstrates the screening of immunomodulatory compounds via fluorescent-based assay. Transgenic mouse-derived T lymphocytes with green fluorescent protein expression cassettes are treated with these compounds. The enhanced and reduced GFP signals enable the identification of the compounds' stimulatory and inhibitory effects, respectively.

A Fluorescence-Based Assay Using Engineered Lymphocytes for Screening Immunomodulatory Compounds

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A protocol for small molecular drug screening based on in-situ synthesis of ultrasmall fluorescent gold nanoclusters (Au NCs) using drug-loaded protein as template is presented. This method is simple to determine the binding affinity of drugs to a target protein by a visible fluorescent signal emitted from the protein-templated Au NCs.

A Rapid and Quantitative Fluorimetric Method for Protein-Targeting Small Molecule Drug Screening

We present in the current study a novel fluorescence-based assay using lymphocytes derived from a transgenic mouse. This assay is suitable for high-throughput screening (HTS) of small molecules endowed with the capacity of either inhibiting or promoting lymphocyte activation.

A Fluorescence-based Lymphocyte Assay Suitable for High-throughput Screening of Small Molecules

This manuscript describes a method to visualize and quantify localized translation events in subcellular compartments. The approach proposed in this manuscript requires a basic confocal imaging system and reagents and is rapid and cost-effective.

Quantitative Immunofluorescence to Measure Global Localized Translation

An easy and adaptable broth microdilution method for screening antifungal compounds and extracts.

Broth Microdilution In Vitro Screening: An Easy and Fast Method to Detect New Antifungal Compounds

Here, we present the detailed procedure of a recently developed protease assay platform utilizing N-terminal hexahistidine/maltose-binding protein and fluorescent protein-fused recombinant substrates attached to the surface of nickel-nitrilotriacetic acid magnetic agarose beads. A subsequent in-gel analysis of the assay samples separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis is also presented.

Use of Recombinant Fusion Proteins in a Fluorescent Protease Assay Platform and Their In-gel Renaturation

Tracking individual translation events allows for high-resolution kinetic studies of cap-dependent translation mechanisms. Here we demonstrate an in vitro single-molecule assay based on imaging interactions between fluorescently labeled antibodies and epitope-tagged nascent peptides. This method enables single-molecule characterization of initiation and peptide elongation kinetics during active in vitro cap-dependent translation.

An In Vitro Single-Molecule Imaging Assay for the Analysis of Cap-Dependent Translation Kinetics

Monitoring changes in the melting temperature of a target protein (i.e., thermal shift assay, TSA) is an efficient method for screening fragment libraries of a few hundred compounds. We present a TSA protocol implementing robotics-assisted nano-Differential Scanning Fluorimetry (nano-DSF) for monitoring intrinsic tryptophan fluorescence and light back-scattering for fragment screening.

Nano-Differential Scanning Fluorimetry for Screening in Fragment-based Lead Discovery

This protocol describes a gel shift biochemical assay for measuring FEN1 (Flap Endonuclease 1) activity and inhibitor development.

Fluorescence-Based Detection of FEN1 Nuclease Activity and Screening of Small-Molecule Inhibitors

Learn a fluorescence-based protocol for screening protein synthesis inhibitors from natural sources. This method supports drug discovery research and provides hands-on training for students in medicinal chemistry.

A Fluorescence-based Protocol for Preliminary Screening of Protein Synthesis Inhibitors from Natural Sources

Explore More Videos