The protocol described here was derived from the article published by Jiang et al. (2020). This work was supported by grants from the MOST (973 Programs nos. 2011CB812502 and 2014CB849902) and by funding support from the Beijing Municipal Government.

All the supplies used in this experiment are listed in the Table of Materials. The step-by-step workflow of the current protocol is shown in Figure 1.
1. Cell culture on sapphire discs
<ol>
	<li>Transfer 3 mm x 0.16 mm sapphire discs to a 2 mL centrifuge tube containing 1 mL of ethanol, and sonicate in an ultrasonic cleaner for 10 min.</li>
	<li>Burn each sapphire disc in an alcohol lamp flame until there are no visible deposits on the surface. 
	NOTE: Through the above method, sapphire discs can be recycled many times.</li>
	<li>Label one side of each sapphire disc with a number using a solvent-resistant pen (Figure 1A). 
	NOTE: The marker pen used needs to be tested to determine whether it is resistant to acetone and methanol solvents in advance to prevent the loss of marks.</li>
	<li>Place the labeled sapphire discs on the bottom of a 35 mm culture dish with the labeled side facing up.</li>
	<li>Sterilize the cell culture dish with sapphire discs under UV light for 30 min.</li>
	<li>Flip the sapphire discs with tweezers (the labeled side facing down), and sterilize under UV light for an additional 30 min. Now, the culture dish containing the sapphire discs is ready for cell culture. 
	NOTE: The sapphire discs can also be sterilized by autoclaving.</li>
	<li>Seed the cells on the sapphire discs prepared above, and grow to 80%-90% confluency in a cell incubator at 37 &#176;C, 95% humidity, and 5% CO2 (Figure 1B). 
	NOTE: Stable HeLa cell lines expressing MTn tags were generated as described in Jiang et al.26.</li>
</ol>
2. High-pressure freezing (HPF)
CAUTION: Liquid nitrogen is used in this experiment. When working with liquid nitrogen, use proper safety procedures and personal protective equipment to prevent frostbite and asphyxiation.
<ol>
	<li>Prepare the high-pressure freezing machine at least 2 h before the experiment according to the manufacturer&#39;s instructions.</li>
	<li>Transfer type A HPF aluminum carriers (recess: 0.025/0.275 mm) to a 2 mL centrifuge tube containing 1 mL of ethanol, and sonicate in an ultrasonic cleaner for 10 min.</li>
	<li>Dry the carriers in a Petri dish covered with qualitative filter paper (medium speed).</li>
	<li>Soak the precleaned carriers in 1-hexadecene. 
	NOTE: BSA is not recommended as a cryoprotectant in the current experiment as it will interfere with the subsequent gold nanoparticle synthesis.</li>
	<li>Use fine tweezers to pick up a sapphire disc with cells from the culture dish; touch the labeled surface with qualitative filter paper (medium speed) to remove the excess medium. 
	NOTE: The thermal conductivity of water is very low, and too much residual water will affect the freezing effect. Retain minimal water to prevent the cells from drying out.</li>
	<li>Mount the sapphire disc into the HPF specimen holder, and quickly cap the disc with a 0.025 mm deep aluminum carrier containing 1-hexadecene (Figure 1C).</li>
	<li>Aspirate excess solution with qualitative filter paper (medium speed).</li>
	<li>Load the specimen holder for high-pressure freezing (Figure 1D). 
	NOTE: The specimen loading process needs to be as fast as possible (within 1 min) to minimize physiological alterations due to changes in the temperature, humidity, pH, and oxygen content.</li>
	<li>Unload the sapphire disc-carrier assembly under liquid nitrogen in a foam cryobox, and store the assembly in a cryovial in liquid nitrogen before use. 
	&#8203;NOTE: The protocol may be paused here. The specimens in cryovials can be stored in a liquid nitrogen dewar for years.</li>
</ol>
3. Freeze-substitution fixation (FSF) and rehydration (Figure 1E)
<ol>
	<li>Fill the automated freeze-substitution machine (AFS) with liquid nitrogen, and cool the chamber to &#8722;90 &#176;C. 
	CAUTION: Liquid nitrogen is used in this experiment. When working with liquid nitrogen, use proper safety procedures and personal protective equipment to prevent frostbite and asphyxiation.</li>
	<li>Prepare 2 mL of 0.01% tannic acid (w/v) in acetone (FSF solution 1) in a 20 mm round polypropylene container (Figure 1E) in a fume hood and freeze it in liquid nitrogen.</li>
	<li>Load the specimens (the sapphire disc-carrier assemblies) into the container with frozen FSF solution 1 under LN2 using a pair of precooled tweezers.</li>
	<li>Transfer the container to the precooled AFS chamber for FSF processing at &#8722;90 &#176;C.</li>
	<li>Keep the specimens at &#8722;90 &#176;C for 1 h; then, separate the sapphire discs from the carriers with precooled tweezers, and make sure the marked sides of the sapphire discs are facing down. 
	NOTE: The tweezers must be sufficiently precooled to prevent temperature changes. The sapphire discs should be easily separated.</li>
	<li>Keep at &#8722;90 &#176;C for a further 8-10 h; then, warm to &#8722;60 &#176;C within 3 h.</li>
	<li>Replace the FSF solution 1 in the container with precooled acetone, and incubate for 1 h. Repeat this acetone wash 2x.</li>
	<li>Warm to &#8722;30 &#176;C within 3 h. During this period, prepare and precool 2 mL of 0.01% uranyl acetate in acetone (FSF solution 2, diluted from 10% uranyl acetate in methanol) in a 2 mL centrifuge tube. 
	CAUTION: Uranyl acetate, used in the current experiment, is radioactive and highly toxic; it must be handled according to proper safety procedures and disposed of as hazardous chemical waste.</li>
	<li>Replace the acetone with FSF solution 2, and incubate at &#8722;30 &#176;C for 3 h.</li>
	<li>Replace the FSF solution 2 in the container with precooled acetone, and incubate for 30 min at &#8722;30 &#176;C. Repeat this acetone wash 2x.</li>
	<li>Warm from &#8722;30 &#176;C to 4 &#176;C within 2 h.</li>
	<li>Replace the acetone with 2 mL of 0.2 M HEPES buffer containing 1 mM CaCl2 and 1 mM MgCl2 at pH 5.5.</li>
	<li>Change the buffer once, and incubate at room temperature for 1 h or at 4 &#176;C overnight. 
	&#8203;NOTE: The protocol may be paused here for one night or continued for at least 6 h until the specimens are frozen again in step 5.</li>
</ol>
4. ANSM-based AuNP synthesis for mammalian cells (Figure 1F)
<ol>
	<li>Wash with PBS-A buffer 3x for 5 min each time.</li>
	<li>Transfer the sapphire discs to a 35 mm culture dish containing 1 mL of PBS-A buffer at room temperature. 
	NOTE: Always keep the marked sides of the sapphire discs facing down, and avoid overlapping the sapphire discs and rubbing off the cells.</li>
	<li>Prepare the reducing solution by adding 4.28 &#181;L of 2-mercaptoethanol into a 2 mL centrifuge tube containing 1 mL of PBS-A buffer and mixing well in a fume hood. 
	CAUTION: 2-Mercaptoethanol is toxic and has a pungent odor; it must be handled according to proper safety procedures.</li>
	<li>Replace the PBS-A buffer in the culture dish with 1 mL of reducing solution, and incubate for 1 h at room temperature.</li>
	<li>Prepare the gold precursor before use.
	<ol>
		<li>Add 80 &#181;L of 10 mM HAuCl4 in ddH2O into a 2 mL centrifuge tube containing 1 mL of reducing solution, and immediately vortex. 
		NOTE: The solution may become cloudy.</li>
		<li>Add 80 &#181;L of 500 mM D-penicillamine in ddH2O into the above solution, and immediately vortex. 
		NOTE: The solution may become clear again.</li>
	</ol>
	</li>
	<li>Replace the solution in the culture dish with the 1 mL of gold precursor solution prepared in step 4.5, and incubate for 2 h at 4 &#176;C. 
	NOTE: One milliliter of the gold precursor is sufficient to react with approximately 1 &#215; 106 cells (confluent on a 35 mm dish). Larger volumes of the precursor are needed when the AuNPs become significantly smaller.</li>
	<li>Weigh 0.0038 g of NaBH4 into a 1.5 mL centrifuge tube, add 1 mL of ice-cold ddH2O, and vortex to make fresh 100 mM NaBH4 just prior to use.</li>
	<li>Add 20-100 &#181;L of 100 mM NaBH4 to the solution from step 4.6, immediately shake to mix well, and incubate for 5 min at room temperature. 
	NOTE: Prepare 100 mM NaBH4 freshly for immediate use. After adding the NaBH4, the color of the solution will gradually turn red and then deepen. If no color change is observed, it largely indicates that the experiment has failed.</li>
	<li>Replace the solution with 2 mL of PBS-A buffer to stop the reaction. 
	NOTE: Stopping the reaction within 30 min is critical, as a prolonged reaction will gradually break down the AuNPs.</li>
</ol>
5. High-pressure freezing and freeze-substitution fixation (Figure 1C-F)
NOTE: The specimens are HPF and FSF again, and the procedure is almost the same as that previously described in section 2 and section 3 with a few modifications.
<ol>
	<li>Prepare 1% osmium tetroxide and 0.1% uranyl acetate in acetone (FSF solution 3, diluted from 4% osmium tetroxide in acetone and 10% uranyl acetate in methanol). 
	CAUTION: Osmium tetroxide and uranyl acetate are highly toxic chemicals; they must be handled according to proper safety procedures and disposed of as hazardous chemical waste.</li>
	<li>Load the specimens (the sapphire disc-carrier assemblies) into the container with frozen FSF solution 3 under LN2 using a pair of precooled tweezers.</li>
	<li>Transfer the container to the precooled AFS chamber for FSF processing at &#8722;90 &#176;C, and run the FSF program as follows: keep at &#8722;90 &#176;C for 8-10 h; then, warm to &#8722;60 &#176;C within 3 h; keep at &#8722;60 &#176;C for 3 h; then, warm to &#8722;30 &#176;C within 3 h; keep at &#8722;30 &#176;C for 3 h; then, warm to 4 &#176;C within 2 h.</li>
	<li>When the temperature reaches 4 &#176;C, transfer the specimens to the fume hood, and maintain at room temperature for 15 min.</li>
	<li>Wash with acetone 3x for 15 min each time.</li>
	<li>Maintain in acetone at room temperature for at least 2 h.</li>
</ol>
6. Resin infiltration, embedding, and polymerization
<ol>
	<li>Prepare the epoxy resin mixture according to the manufacturer&#39;s instructions before use. 
	CAUTION: The epoxy resin used in the current experiment is toxic prior to polymerization; it must be handled according to proper safety procedures. Unpolymerized resin should be disposed of as hazardous chemical waste or polymerized prior to disposal.</li>
	<li>Transfer the sapphire discs to flat-bottom embedding capsules, and infiltrate with resin for at least 30 min at room temperature. 
	NOTE: Keep the marked sides of the sapphire discs facing down.</li>
	<li>Polymerize the specimen at 60 &#176;C in an oven for at least 18 h. 
	&#8203;NOTE: The protocol can be paused here after polymerization. Polymerized specimen blocks can be stored in a dry container for years.</li>
</ol>
7. Ultrathin sectioning
<ol>
	<li>Trim the polymerized specimen blocks carefully using a razor to expose the sapphire discs.</li>
	<li>Dip the block tips with sapphire discs into liquid nitrogen for several seconds, and thaw at room temperature. Repeat the freeze-thaw action several times to detach the discs from the blocks. 
	NOTE: Avoid prolonged freezing, as this may crack the specimen blocks. Gently detach the sapphire discs with a blade, avoiding excessive force, which may damage the samples.</li>
	<li>Trim the block face to a trapezoidal shape for ultrathin-sectioning (Figure 1G).</li>
	<li>Obtain 70-100 nm ultrathin sections with an ultramicrotome (Figure 1H).</li>
	<li>Pick the sections on 200-mesh hexagonal copper grids. 
	&#8203;NOTE: The protocol can be paused here after sectioning. The sections on girds can be stored in a dry container for years. If desired, the sections on grids can be stained with 2% uranyl acetone to obtain better membrane contrast. Lead staining should be avoided as it will mask the nanogold particle signals.</li>
</ol>
8. TEM imaging (Figure 1I)
<ol>
	<li>Examine the ultrathin sections on copper grids using a transmission electron microscope. Typically, a magnification range from 11 kX to 30 kX is suitable for visualizing AuNPs in cells, and an appropriate defocus value is needed to ensure 2-3 nm AuNPs are visible.</li>
</ol>

Direct Synthesis of Gold Nanoparticles for Protein Localization Analysis

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The author declares no conflicts of interest.

The study presents here a robust clonable EM labeling technology for the single-molecule visualization of protein molecules within the cellular environment with ultrastructural resolution. The AuNPs directly synthesized on genetically encoded cysteine-rich tags provide unambiguous and precise localization of the target proteins. High-pressure freezing and freeze-substitution technique excellently preserve the ultrastructure of biological samples. Taken together, the clonable electron microscopy labeling technology presen...

In the postgenomic era, the development of green fluorescent protein (GFP) as a single-molecule reporter for light microscopy has revolutionized the field of modern life science research1,2. For decades, electron microscopy (EM) has been a powerful tool for intuitively observing the cellular ultrastructure with nanoscale resolution3; however, the precise identification and localization of protein molecules remain challenging.
The most commonly used EM labeling technique is the immunoelectron microscopy (IEM) labeling technique, which is based on the antigen-antibody reaction. However, although many techniques have been developed in the field of IEM labeling, including pre-embedding IEM and post-embedding IEM (on resin sections or hydrated cryosections), it still suffers from low labeling efficiency (&#60;10%)4,5, which is related to the sample preparation and antibody quality. To overcome these limitations, developing genetically encoded tags has great application potential.
Two main types of EM tags have been thoroughly explored in recent years. One type is the DAB staining method, which utilizes tags such as APEX2 to oxidize 3,3&#39;-diaminobenzidine (DAB) to osmiophilic polymers for EM visualization6,7,8,9,10,11,12. It enables the labeling of high-abundance proteins in subcellular regions but is not suitable for single-molecule counting. The other type utilizes metal-binding proteins, such as ferritin13 and metallothionein (MT)14,15,16,17,18,19,20,21,22,23,24,25,26, to generate electron-dense metal deposits in situ for EM visualization. Only the latter has real potential for single-molecule visualization and counting. The molecular size of ferritin is too large (~450 kD) for it to be used as a promising tag, whereas the small size (~5 kD) of MT and its ability to bind various ions through its 20 cysteines have attracted great attention. Several labs have tried to label purified MT-fusion proteins or MT-expressing cells by incubating directly with Au+. These attempts have initially proved that MT tags can bind gold ions to form high-contrast signals, but none have really achieved the effective identification of individual proteins in cells, and they are not widely applicable14,15,16,17,18,19,20,21,22,23.
The ANSM-based AuNP synthesis technique, which involves synthesizing 2-6 nm-sized AuNPs directly on cysteine-rich tags (e.g., MT, the MT variants MTn and MT&#945;, AFP) as electron-dense labels for EM visualization, is the first reliable and applicable approach for protein labeling and single molecule detection in cells24,25,26. It allows for the specific synthesis of AuNPs on isolated tag fusion proteins and has achieved an unprecedented labeling efficiency in nonfixed or chemically fixed prokaryotic (E. coli) and eukaryotic (S. pombe) cells. However, implementing the same protocol in more advanced systems such as mammalian cells or even tissues involves additional challenges, such as the more complex intracellular redox homeostasis and more fragile cellular structure.
This study presents a clonable EM labeling technology, which combines the novel ANSM-based AuNP synthesis technique for labeling genetically encoded cysteine-rich tags (MT) with the HPF/FSF-rehydration-HPF/FSF sample preparation method, enables the unambiguous single-molecule identification of tagged proteins in the ER membrane, ER lumen, and mitochondrial matrix in HeLa cells. The current method combines the characteristics of high labeling efficiency, a high signal-to-noise ratio, single-molecule labeling, and strong universality, and this method has broad application prospects in life science research.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>0.025 mm/0.275 mm Aluminum carrier</td>
			<td>Beijing Wulundes Biotech Ltd., or Engineering Office of M. Wohlwend</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>0.2 M HEPES buffer</td>
			<td></td>
			<td></td>
			<td>Dissolve HEPES (0.2 M) in 980 mL of ddH2O, then add 10 mL of 100 mM MgCl2 and 10 mL of 100 mM CaCl2 (final concentration 1 mM), respectively, adjust pH to 5.5</td>
		</tr>
		<tr>
			<td>1.5 mL MaxyClear snaplock microtube</td>
			<td>Axygen Scientific</td>
			<td>MCT150C</td>
			<td></td>
		</tr>
		<tr>
			<td>2 mL polypropylene screw cap microtubes</td>
			<td>Biologix</td>
			<td>81-0204</td>
			<td></td>
		</tr>
		<tr>
			<td>200 mesh hexagonal copper grid</td>
			<td>Tedpella inc</td>
			<td>G200HEX</td>
			<td></td>
		</tr>
		<tr>
			<td>2-mercaptoethanol</td>
			<td>Amresco</td>
			<td>0482-250ML</td>
			<td></td>
		</tr>
		<tr>
			<td>35 mm cell culture dishes</td>
			<td>Corning</td>
			<td>430165</td>
			<td></td>
		</tr>
		<tr>
			<td>50 mL polypropylene centrifuge tubes</td>
			<td>Corning</td>
			<td>430928</td>
			<td></td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Beiijng Tong Guang Fine Chemicals Company</td>
			<td>31025</td>
			<td></td>
		</tr>
		<tr>
			<td>Automated freeze substitution machine</td>
			<td>Leica</td>
			<td>AFS2</td>
			<td></td>
		</tr>
		<tr>
			<td>Customized 3.05 mm x 0.66 mm specimen holders for HPF</td>
			<td>Beijing Wulundes Biotech Ltd.</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>D-penicillamine</td>
			<td>TCI</td>
			<td>P0147</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#8217;s modified Eagle medium</td>
			<td>GBICO</td>
			<td>C11965500BT</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>GBICO</td>
			<td>10099-141C</td>
			<td></td>
		</tr>
		<tr>
			<td>Flat bottom embedding capsule</td>
			<td>Tedpella inc</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Foam cryobox</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Formvar 15/95 resin</td>
			<td>Electron Microscopy Sciences</td>
			<td>15800</td>
			<td></td>
		</tr>
		<tr>
			<td>HAuCl4</td>
			<td>Sigma</td>
			<td>4022-1G</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>sigma&#160;</td>
			<td>H3375-500G</td>
			<td></td>
		</tr>
		<tr>
			<td>HPF machine</td>
			<td>Wohlwend&#160;</td>
			<td>HPF compact01</td>
			<td></td>
		</tr>
		<tr>
			<td>Methonal&#160;</td>
			<td>Beiijng Tong Guang Fine Chemicals Company</td>
			<td>12397</td>
			<td></td>
		</tr>
		<tr>
			<td>NaBH4</td>
			<td>Sigma</td>
			<td>480886-25G</td>
			<td></td>
		</tr>
		<tr>
			<td>OsO4</td>
			<td>Electron Microscopy Sciences</td>
			<td>19110</td>
			<td></td>
		</tr>
		<tr>
			<td>PBS-A buffer</td>
			<td></td>
			<td></td>
			<td>Dissolve NaH2PO4 (1.125 mM), Na2HPO4 (3.867 mM), NaCl (100 mM) in 1 L of ddH2O, adjust to pH 7.4</td>
		</tr>
		<tr>
			<td>Qualitative filter paper (medium speed)</td>
			<td>Beyotime Biotechnology</td>
			<td>FFT08</td>
			<td></td>
		</tr>
		<tr>
			<td>Sapphire discs</td>
			<td>Beijing Wulundes Biotech Ltd., or Engineering Office of M. Wohlwend</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Solvent resistant pen</td>
			<td>Electron Microscopy Sciences</td>
			<td>62053-B</td>
			<td></td>
		</tr>
		<tr>
			<td>SPI-Pon 812 resin</td>
			<td>SPI Inc</td>
			<td>02659-AB</td>
			<td></td>
		</tr>
		<tr>
			<td>Transmission electron microscopy</td>
			<td>FEI</td>
			<td>Tecnai G2 spirit</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>GBICO</td>
			<td>C25200-056</td>
			<td></td>
		</tr>
		<tr>
			<td>Tweezers</td>
			<td>Dumont</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Ultramictotome</td>
			<td>Leica</td>
			<td>FC7</td>
			<td></td>
		</tr>
		<tr>
			<td>Uranyl acetate</td>
			<td>Electron Microscopy Sciences</td>
			<td>22400</td>
			<td></td>
		</tr>
	</tbody>
</table>

direct synthesis of em-visible gold nanoparticles in cells for protein localization analysis with well-preserved ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various physiological or pathological processes in all living organisms. Therefore, the development of clonable tags that can be used as electron microscopy probes is of great value, just as fluorescent proteins have played a crucial role in the field of optical imaging. The autonucleation suppression mechanism (ANSM) was recently uncovered, which allows for the specific synthesis of gold nanoparticles (AuNPs) on cysteine-rich tags, such as metallothionein (MT) and antifreeze protein (AFP).
Based on the ANSM, an electron microscopy labeling technology was developed, which enables the specific detection of tagged proteins in prokaryotic and eukaryotic cells with an unprecedented labeling efficiency. This study illustrates a protocol for the detection of MTn (an engineered MT variant lacking aldehyde-reactive residues) fusion proteins in mammalian cells with well-preserved ultrastructure. In this protocol, high-pressure freezing and freeze-substitution fixation were performed using non-aldehyde fixatives (such as tannic acid, uranyl acetate) to preserve near-native ultrastructure and avoid damage to the tag activity caused by aldehyde crosslinking.
A simple one-step rehydration was used prior to the ANSM-based AuNP synthesis. The results showed that the tagged proteins targeted various organelles, including the membranes and the lumen of the endoplasmic reticulum (ER), and mitochondrial matrices were detected with high efficiency and specificity. This research provides biologists with a robust protocol to address an enormous range of biological questions at the single-molecule level in cellular ultrastructural contexts.

The ANSM-based AuNP synthesis technique is an extremely useful tool for labeling and detecting MT-tagged proteins with TEM26. To validate its robustness in mammalian cells, three stable cell lines expressing EGFP-MTn-KDEL, Ost4-EGFP-MTn, or Mito-acGFP-MTn in Hela cells were generated. KDEL is a canonical C-terminal endoplasmic reticulum (ER) retention/retrieval sequence, which maintains the fusion protein EGFP-MTn-KDEL within the ER lumen or the perinuclear space of the nuclear envelope (NE). Ost4...

Watch this Scientific Journal Video about Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure at JoVE.com

Author Spotlight: Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure  

The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique.

Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure

Analyzing the precise localization of protein molecules in cells with ultrastructural resolution is of great significance for the study of various ...

direct-synthesis-em-visible-gold-nanoparticles-cells-for-protein

Research

JoVE Journal

Biology

1.3K Views.  National Institute of Biological Sciences, Beijing. The present protocol describes a clonable electron microscopy labeling technology for detecting metallothionein-tagged proteins in cells using a novel autonucleation suppression mechanism-based gold nanoparticle synthesis technique. 

Video: Author Spotlight: Direct Synthesis of EM-Visible Gold Nanoparticles in Cells for Protein Localization Analysis with Well-Preserved Ultrastructure  

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields of regenerative medicine, basic cell biology, treatment of diseases, and reprogramming of cells. Here, we describe a step by step protocol for generation of modified mRNA with reduced immune activation potential and increased stability, quality control of produced mRNA, transfection of cells with mRNA and verification of the induced protein expression by flow cytometry. Up to 3 days after a single transfection with eGFP mRNA, the transfected HEK293 cells produce eGFP. In this video article, the synthesis of eGFP mRNA is described as an example. However, the procedure can be applied for production of other desired mRNA. Using the synthetic modified mRNA, cells can be induced to transiently express the desired proteins, which they normally would not express.

In Vitro Synthesis of Modified mRNA for Induction of Protein Expression in Human Cells

In this video article, we describe the in vitro synthesis of modified mRNA for induction of protein expression in cells.

The exogenous delivery of coding synthetic messenger RNA (mRNA) for induction of protein synthesis in desired cells has enormous potential in the fields ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...

A Graphical User Interface for Software-assisted Tracking of Protein Concentration in Dynamic Cellular Protrusions

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex signaling mechanisms underlying filopodial initiation, elongation and subsequent stabilization or retraction, it is crucial to determine the spatio-temporal protein activity in these dynamic structures. To analyze protein function in filopodia, we recently developed a semi-automated tracking algorithm that adapts to filopodial shape-changes, thus allowing parallel analysis of protrusion dynamics and relative protein concentration along the whole filopodial length. Here, we present a detailed step-by-step protocol for optimized cell handling, image acquisition and software analysis. We further provide instructions for the use of optional features during image analysis and data representation, as well as troubleshooting guidelines for all critical steps along the way. Finally, we also include a comparison of the described image analysis software with other programs available for filopodia quantification. Together, the presented protocol provides a framework for accurate analysis of protein dynamics in filopodial protrusions using image analysis software.

We present a software solution for semi-automated tracking of relative protein concentration along the length of dynamic cellular protrusions.

Filopodia are dynamic, finger-like cellular protrusions associated with migration and cell-cell communication. In order to better understand the complex ...

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These age-associated disorders are characterized by the appearance of protein aggregates with fibrillary structure in the brains of these patients. Exactly why normally soluble proteins undergo an aggregation process remains poorly understood. The discovery that protein aggregation is not limited to disease processes and instead part of the normal aging process enables the study of the molecular and cellular mechanisms that regulate protein aggregation, without using ectopically expressed human disease-associated proteins. Here we describe methodologies to examine inherent protein aggregation in Caenorhabditis elegans through complementary approaches. First, we examine how to grow large numbers of age-synchronized C. elegans to obtain aged animals and we present the biochemical procedures to isolate highly-insoluble-large aggregates. In combination with a targeted genetic knockdown, it is possible to dissect the role of a gene of interest in promoting or preventing age-dependent protein aggregation by using either a comprehensive analysis with quantitative mass spectrometry or a candidate-based analysis with antibodies. These findings are then confirmed by in vivo analysis with transgenic animals expressing fluorescent-tagged aggregation-prone proteins. These methods should help clarify why certain proteins are prone to aggregate with age and ultimately how to keep these proteins fully functional.

Methods to Study Changes in Inherent Protein Aggregation with Age in Caenorhabditis elegans

The goal of the method presented here is to explore protein aggregation during normal aging in the model organism C. elegans. The protocol represents a powerful tool to study the highly insoluble large aggregates that form with age and to determine how changes in proteostasis impact protein aggregation.

In the last decades, the prevalence of neurodegenerative disorders, such as Alzheimer's disease (AD) and Parkinson's disease (PD), has grown. These ...

The CryoAPEX Method for Electron Microscopy Analysis of Membrane Protein Localization Within Ultrastructurally-Preserved Cells

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise subcellular localization of proteins is thus important for answering many biological questions. The quest for a robust label to identify protein localization combined with adequate cellular preservation and staining has been historically challenging. Recent advances in electron microscopy (EM) imaging have led to the development of many methods and strategies to increase cellular preservation and label target proteins. A relatively new peroxidase-based genetic tag, APEX2, is a promising leader in cloneable EM-active tags. Sample preparation for transmission electron microscopy (TEM) has also advanced in recent years with the advent of cryofixation by high pressure freezing (HPF) and low-temperature dehydration and staining via freeze substitution (FS). HPF and FS provide excellent preservation of cellular ultrastructure for TEM imaging, second only to direct cryo-imaging of vitreous samples. Here we present a protocol for the cryoAPEX method, which combines the use of the APEX2 tag with HPF and FS. In this protocol, a protein of interest is tagged with APEX2, followed by chemical fixation and the peroxidase reaction. In place of traditional staining and alcohol dehydration at room temperature, the sample is cryofixed and undergoes dehydration and staining at low temperature via FS. Using cryoAPEX, not only can a protein of interest be identified within subcellular compartments, but also additional information can be resolved with respect to its topology within a structurally preserved membrane. We show that this method can provide high enough resolution to decipher protein distribution patterns within an organelle lumen, and to distinguish the compartmentalization of a protein within one organelle in close proximity to other unlabeled organelles. Further, cryoAPEX is procedurally straightforward and amenable to cells grown in tissue culture. It is no more technically challenging than typical cryofixation and freeze substitution methods. CryoAPEX is widely applicable for TEM analysis of any membrane protein that can be genetically tagged.

This protocol describes the cryoAPEX method, in which an APEX2-tagged membrane protein can be localized by transmission electron microscopy within optimally-preserved cell ultrastructure.

Key cellular events like signal transduction and membrane trafficking rely on proper protein location within cellular compartments. Understanding precise ...

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and degradation instigates a multitude of age-related diseases. Decline of proteostasis quality control mechanisms is a hallmark of ageing. Biochemical methods to detect de novo protein synthesis are still limited, have several disadvantages and cannot be performed in live cells or animals. Caenorhabditis elegans, being transparent and easily genetically modified, is an excellent model to monitor protein synthesis rates by using imaging techniques. Here, we introduce and describe a method to measure de novo protein synthesis in vivo utilizing fluorescence recovery after photobleaching (FRAP). Transgenic animals expressing fluorescent proteins in specific cells or tissues are irradiated by a powerful light source resulting in fluorescence photobleaching. In turn, assessment of fluorescence recovery signifies new protein synthesis in cells and/or tissues of interest. Hence, the combination of transgenic nematodes, genetic and/or pharmacological interventions together with live imaging of protein synthesis rates can shed light on mechanisms mediating age-dependent proteostasis collapse.

Assessment of de novo Protein Synthesis Rates in Caenorhabditis elegans

Here, we introduce and describe a nonradioactive and noninvasive method to assess de novo protein synthesis in vivo, utilizing the nematode Caenorhabditis elegans and fluorescence recovery after photobleaching (FRAP). This method can be combined with genetic and/or pharmacological screens to identify novel modulators of protein synthesis.

Maintaining a healthy proteome is essential for cell and organismal homeostasis. Perturbation of the balance between protein translational control and ...

Tethered Bilayer Lipid Membranes to Monitor Heat Transfer between Gold Nanoparticles and Lipid Membranes

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry using tethered bilayer lipid membranes (tBLMs) assembled on gold electrodes. Irradiated modified GNPs, such as streptavidin-conjugated GNPs, are embedded in tBLMs containing target molecules, such as biotin. By using this approach, the heat transfer processes between irradiated GNPs and model bilayer lipid membrane with entities of interest are mediated by a horizontally focused laser beam. The thermal predictive computational model is used to confirm the electrochemically induced conductance changes in the tBLMs. Under the specific conditions used, detecting heat pulses required specific attachment of the gold nanoparticles to the membrane surface, while unbound gold nanoparticles failed to elicit a measurable response. This technique serves as a powerful detection biosensor which can be directly utilized for the design and development of strategies for thermal therapies that permits optimization of the laser parameters, particle size, particle coatings and composition.

This work outlines a protocol to achieve dynamic, non-invasive monitoring of heat transfer from laser-irradiated gold nanoparticles to tBLMs. The system combines impedance spectroscopy for the real-time measurement of conductance changes across the tBLMs, with a horizontally focused laser beam that drives gold nanoparticle illumination, for heat production.

Here we report a protocol to investigate the heat transfer between irradiated gold nanoparticles (GNPs) and bilayer lipid membranes by electrochemistry ...

Embedding CFPS Reactions in a Variety of Hydrogel Matrices

Methods for Embedding Cell-Free Protein Synthesis Reactions in Macro-Scale Hydrogels

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. While the dominant paradigm for the deployment of gene networks is within a cellular chassis, synthetic gene networks may also be deployed in cell-free environments. Promising applications of cell-free gene networks include biosensors, as these devices have been demonstrated against biotic (Ebola, Zika, and SARS-CoV-2 viruses) and abiotic (heavy metals, sulfides, pesticides, and other organic contaminants) targets. Cell-free systems are typically deployed in liquid form within a reaction vessel. Being able to embed such reactions in a physical matrix, however, may facilitate their broader application in a wider set of environments. To this end, methods for embedding cell-free protein synthesis (CFPS) reactions in a variety of hydrogel matrices have been developed. One of the key properties of hydrogels conducive to this work is the high-water reconstitution capacity of hydrogel materials. Additionally, hydrogels possess physical and chemical characteristics that are functionally beneficial. Hydrogels can be freeze-dried for storage and rehydrated for use later. Two step-by-step protocols for the inclusion and assay of CFPS reactions in hydrogels are presented. First, a CFPS system can be incorporated into a hydrogel via rehydration with a cell lysate. The system within the hydrogel can then be induced or expressed constitutively for complete protein expression through the hydrogel. Second, cell lysate can be introduced to a hydrogel at the point of polymerization, and the entire system can be freeze-dried and rehydrated at a later point with an aqueous solution containing the inducer for the expression system encoded within the hydrogel. These methods have the potential to allow for cell-free gene networks that confer sensory capabilities to hydrogel materials, with the potential for deployment beyond the laboratory.

Author Spotlight: A Novel Approach for Embedding Cell-Free Protein Synthesis Reactions in Hydrogels 

Here, we present two protocols for embedding cell-free protein synthesis reactions in macro-scale hydrogel matrices without the need for an external liquid phase.

Synthetic gene networks provide a platform for scientists and engineers to design and build novel systems with functionality encoded at a genetic level. ...