Name: fluorescent silver staining of proteins in polyacrylamide gels
Uploaded: 2019-04-21T13:00:00
Description: Watch this Scientific Journal Video about Fluorescent Silver Staining of Proteins in Polyacrylamide Gels at JoVE.com

The authors would like to thank Patrick Chan at the Ming Wai Lau Centre for Reparative Medicine, Karolinska Institutet, for his technical support. S. X. is grateful for the support from the Swedish Research Council (Grant No. 2017-06344).

1. Preparation of the Gel
Note: The demonstration follows a standard protocol to prepare the gel for staining shortly after SDS-PAGE12. In brief, the following steps describe the preparation of the samples and gel electrophoresis.
<ol>
	<li>Perform SDS-PAGE with 4% - 12% Bis-Tris protein gels (1 mm, 15-well) using a mini gel tank filled with 2-(N-morpholino)ethanesulfonic acid (MES) buffer.</li>
	<li>Dilute the samples with a mixture of distilled water, lithi...

<ol>
	<li>Chevalier, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Highlights+on+the+capacities+of+&amp;#34;Gel-based&amp;#34;+proteomics.">Highlights on the capacities of &amp;#34;Gel-based&amp;#34; proteomics.</a> Proteome Science. 8 (1), (2010).</li><li>Harris, L. R., Churchward, M. A., Butt, R. H., Coorssen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Assessing+detection+methods+for+gel-based+proteomic+analyses.">Assessing detection methods for gel-based proteomic analyses.</a> Journal of Proteome Research. 6 (4), 1418-1425 (2007).</li><li>Gauci, V. J., Wright, E. P., Coorssen, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+proteomics:+assessing+the+spectrum+of+in-gel+protein+detection+methods.">Quantitative proteomics: assessing the spectrum of in-gel protein detection methods.</a> Journal of Chemical Biology. 4 (1), 3-29 (2011).</li><li>Candiano, G., Bruschi, 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=Blue+silver:+A+very+sensitive+colloidal+Coomassie+G-250+staining+for+proteome+analysis.">Blue silver: A very sensitive colloidal Coomassie G-250 staining for proteome analysis.</a> Electrophoresis. 25 (9), 1327-1333 (2004).</li><li>Chevallet, M., Luche, S., Rabilloud, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+staining+of+proteins+in+polyacrylamide+gels.">Silver staining of proteins in polyacrylamide gels.</a> Nature Protocols. 1 (4), 1852-1858 (2006).</li><li>Jin, L. T., Hwang, S. Y., Yoo, G. S., Choi, J. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Sensitive+silver+staining+of+protein+in+sodium+dodecyl+sulfate-polyacrylamide+gels+using+an+azo+dye,+calconcarboxylic+acid,+as+a+silver-ion+sensitizer.">Sensitive silver staining of protein in sodium dodecyl sulfate-polyacrylamide gels using an azo dye, calconcarboxylic acid, as a silver-ion sensitizer.</a> Electrophoresis. 25 (15), 2494-2500 (2004).</li><li>Celis, J. 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=.">.</a> Cell Biology: A Laboratory Handbook. , (2006).</li><li>Bartsch, H., Arndt, C., Koristka, S., Cartellieri, M., Bachmann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver+Staining+Techniques+of+Polyacrylamide+Gels.">Silver Staining Techniques of Polyacrylamide Gels.</a> Methods in Molecular Biology. 869 (1), 481-486 (2012).</li><li>Rabilloud, T., Vuillard, L., Gilly, C., Lawrence, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Silver-staining+of+proteins+in+polyacrylamide+gels:+a+general+overview.">Silver-staining of proteins in polyacrylamide gels: a general overview.</a> Cellular and Molecular Biology. 40 (1), 57-75 (1994).</li><li>Zhou, M., Yu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Proteomic+Analysis+by+Two-Dimensional+Polyacrylamide+Gel+Electrophoresis.">Proteomic Analysis by Two-Dimensional Polyacrylamide Gel Electrophoresis.</a> Advances in Protein Chemistry. 65 (1), 57-84 (2003).</li><li>Xie, S., 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=Fluorogenic+Ag+-Tetrazolate+Aggregation+Enables+Efficient+Fluorescent+Biological+Silver+Staining.">Fluorogenic Ag+-Tetrazolate Aggregation Enables Efficient Fluorescent Biological Silver Staining.</a> Angewandte Chemie International Edition. 57 (20), 5750-5753 (2018).</li><li>. Protein gel electrophoresis technical handbook Available from: <a target="_blank" href="https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-gel-electrophoresis-technical-handbook.pdf">https://www.thermofisher.com/content/dam/LifeTech/global/Forms/PDF/protein-gel-electrophoresis-technical-handbook.pdf</a> (2015)</li></ol>

Presented here is a novel fluorescent silver staining method for proteins in polyacrylamide gels. This strategy integrates conventional silver stains and fluorescent stains. The staining exploits the selective binding of silver ion to proteins as in other silver stains but employs a highly sensitive fluorogenic silver probe TPE-4TA to light up the silver bound proteins. Since the fluorogenic probe TPE-4TA can sense silver ions at a fairly low concentration in the nanomolar range<sup clas...

Many staining methods have been used to visualize proteins following gel electrophoresis, for example using colorimetric dyes such as Coomassie Brilliant Blue, silver stain, fluorescence, or radioactive labeling1,2,3,4. Silver staining is considered to be one of the most sensitive techniques for protein detection requiring simple and cheap reagents. It can be categorized into two major families: the ammoniacal silver stain and the silver nitrate stain5,6. In the alkaline ammoniacal silver method, the silver-diamine complex is produced with ammonia and sodium hydroxide and reduced to metallic silver during development using an acidic formaldehyde solution. The stain accommodates efficiently for basic proteins but shows a compromised performance for acidic and neutral proteins and is, furthermore, limited to classical glycine and taurine electrophoretic systems. In comparison, the silver nitrate stains exploit the high bio-affinity of silver ions to protein, primarily the sulfhydryl and carboxyl groups from the side chains, and tend to stain acidic proteins more efficiently7. After silver ion binding, a developing solution (typically made of a metal carbonate solution containing formaldehyde and sodium thiosulphate) is applied to reduce silver ions to metallic silver grains, which build up a brown-to-dark color to visualize the protein bands.
Although silver staining has been well known for its versatility and high sensitivity since its development in the 1970s8, the method is frequently regarded as tricky. Silver-staining methods have time-restricted steps and show low reproducibility. Since the color of silver stain is usually not uniform and dependent on the reduction step, which is hard to control, the silver stain is not a quantitative method and, thus, not recommended for gel comparison study and protein quantification9. Methods optimized in sensitivity may utilize aldehydes which can also provide a more uniform staining10. However, this is at the expense of further downstream analysis due to the crosslinking of proteins by aldehydes. Fast protocols mostly combine or shorten steps to reduce time, compromising the reproducibility and uniformity of the stain5. As a result, there are numerous silver staining variants within protein gel staining, each optimized to suit certain requirements; for example, simplicity, sensitivity, or peptide recovery rate for downstream analysis. These attributes may also have an impact on each other, and satisfying all requirements in one protocol can be difficult.
In this work, we introduce a new fluorescent silver staining method for protein detection in polyacrylamide gel. In this method, we use a fluorogenic probe for silver ions, TPE-4TA (Figure 1), to visualize the silver-impregnated proteins11. TPE-4TA is designed by the aggregation-induced emission (AIE) principle. It is non-emissive when dissolved in aqueous solution, but is highly emissive in the presence of silver ions. By replacing the chromogenic development in traditional silver stains with a fluorogenic developing step, the fluorescent silver method enables the robust staining of total proteins with a reduced background.
Furthermore, the fluorescent silver stain showed a good dynamic linear range for protein quantification, which is comparable with the widely-used SYPRO Ruby stain and not achievable with traditional silver stains. The gel can be imaged on commonly used gel documentation systems with an ultraviolet lamp (excitation wavelength: 302/365 nm channel; emission: ~490 - 530 nm) at many biological labs.

<table>
	<tbody>
		<tr>
			<td> 
			LDS Sample Buffer (4X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0007</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td> 
			4-12% Bis-Tris Protein Gels, 1.0 mm, 15-well</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0323BOX</td>
			<td>Precast gel</td>
		</tr>
		<tr>
			<td>Sample Reducing Agent (10X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0004</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>MES SDS Running Buffer (20X)</td>
			<td>Thermo Fisher Scientific</td>
			<td>NP0002</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Mini Gel Tank</td>
			<td>Thermo Fisher Scientific</td>
			<td>A25977</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>300W Power Supply (230 VAC)</td>
			<td>Thermo Fisher Scientific</td>
			<td>PS0301</td>
			<td>Equipment</td>
		</tr>
		<tr>
			<td>Unstained Protein Ladder</td>
			<td>Thermo Fisher Scientific</td>
			<td>26614</td>
			<td>Sample</td>
		</tr>
		<tr>
			<td>Silver nitrate</td>
			<td>Sigma-Aldrich</td>
			<td>31630-25G-R</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Ethanol</td>
			<td>Bragg and co.</td>
			<td>42520J</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Acetic acid</td>
			<td>J.T. Baker</td>
			<td>103201A</td>
			<td>Reagent</td>
		</tr>
		<tr>
			<td>Milli-Q Synthesis A10</td>
			<td>Merk</td>
			<td>-</td>
			<td>Provides 18.2 M&#937;.cm water</td>
		</tr>
		<tr>
			<td>gel documentation system (c600 model)</td>
			<td>Azure biosystems</td>
			<td>-</td>
			<td>Equipment</td>
		</tr>
	</tbody>
</table>

fluorescent silver staining of proteins in polyacrylamide gels

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

The protein bands stained by the fluorescent silver stain exhibit an intense green fluorescence under a 365 nm UV lamp. All 14 protein bands (10 - 200 kDa), from top to bottom, were clearly visible, correlating well with the 14 red-colored ones stained by the SYPRO Ruby dye (Figure 2)10.
Regarding quantitative protein detection, the gels were imaged by a gel imaging system us...

Watch this Scientific Journal Video about Fluorescent Silver Staining of Proteins in Polyacrylamide Gels at JoVE.com

Fluorescent Silver Staining of Proteins in Polyacrylamide Gels

Here, we describe a detailed protocol outlining a new fluorescent staining technique for total protein detection in polyacrylamide gels. The protocol utilizes a silver ion-specific fluorescence turn-on probe, which detects Ag+-protein complexes, and eliminates certain limitations of traditional chromogenic silver stains.

Silver staining is a colorimetric technique widely used to visualize protein bands in polyacrylamide gels following sodium dodecyl sulfate-polyacrylamide ...

This is a novel gel-staining method that utilizes a fluorogenic silver probe to convert the traditional silver staining into a fluorescent silver staining. This method showed improved performance compared with conventional silver nitrate staining and SYPRO Ruby staining while using a simple and straightforward protocol without aldehydes. Demonstrating the procedure will be Alex Wong, a research assistant from my laboratory.To begin this procedure, dilute the protein samples with a solution containing distilled water, LDS buffer, and a sample-reducing agent. Set up a mini gel tank filled with MES buffer to perform SDS-PAGE with a 4-12%Bis-Tris protein gel. Load the wells with the prepared protein sample, then run the gel at a constant voltage of 200 volts for 30 minutes.After electrophoresis, submerge the gel in a 100-milliliter solution containing ethanol and acetic acid on an orbital shaker and incubate twice for 30 minutes each at room temperature while shaking at 50 rpm. Next, wash the gel three times with ultrapure water in a clean container with each wash lasting 10 minutes. First, dissolve 0.01 grams of silver nitrate in 10 milliliters of ultrapure water to prepare a stock solution with a concentration of 0.1%Add 100 microliters of the stock solution into 100 milliliters of ultrapure water to make the silver nitrate working solution.In a fume hood, submerge the gel in 100 milliliters of the silver nitrate working solution in a sealed glass chamber. Use aluminum foil to protect the gel from light during impregnation and incubate for one hour while shaking at 50 rpm on an orbital shaker. After this, wash the gel twice with ultrapure water in a clean container, with each wash using approximately 100 milliliters of water and lasting 60 seconds.It is important to use ultrapure water to clean the gel after the silver impregnation step to minimize background staining. First, add 50 milliliters of ultrapure water to three milligrams of TPE-4TA dye. Sonicate the solution for three minutes and add five microliters of one-molar sodium hydroxide solution in between each sonication session to help dissolve the dye, usually up to three times.Then check the fluorescence of the solution under a 365-nanometer UV lamp to ensure that the dye is fully dissolved. Only weak or non-emissive solutions indicate full dissolution. To prepare the fluorogenic developing solution, add 10 milliliters of the TPE-4TA stock solution to 90 milliliters of ultrapure water.Use a pH meter to check the pH of the solution. Tune the solution to a pH between 7 and 9, using diluted sodium hydroxide solution or acetic acid if the pH is out of range. Next transfer the gel to a clean and sealable container with 100 milliliters of the fluorogenic developing solution and ensure that the gel is completely immersed.Seal the container and cover it to protect it from light. Shake the container overnight on an orbital shaker at 50 rpm and at room temperature. Transfer the gel to a clean container and destain it in 100 milliliters of 10%ethanol for 30 minutes.Then rinse the gel in ultrapure water for five minutes. The gel can be visualized on a benchtop transilluminator or imaged on a gel documentation machine at the 365-nanometer channel or the 302-nanometer channel. In this study, a novel fluorescent silver staining method is used to stain proteins in polyacrylamide gels.The protein bands stained by the fluorescent silver stain exhibit an intense green under a 365-nanometer UV lamp. All 14 protein bands are clearly visible and correlate well with the red-colored bands stained by the SYPRO Ruby dye. This fluorescent silver staining method appears to have a high resolution.For some protein bands, the sensitivity of the fluorescent silver stain is also slightly better than that of the fluorescent SYPRO Ruby stain. In particular the performance of this stain is improved for the protein bands between 10 and 40 kilodaltons, which indicates that the new method is particularly useful for the detection of proteins with a low molecular weight. As can be seen, the data also suggests that the fluorescent silver stain gives a good and uniform linearity for all 14 proteins over a relatively broad range of protein quantification.While the silver nitrate stain gave a high level of background signal and distorted peaks, the fluorescent silver stain detected the bands with good contrast and uniform intensity distribution comparable to the SYPRO Ruby stain across all 14 proteins. Washing steps are important as they limit the residual fixation solution from disrupting silver impregnation or fluorogenic development. When using silver nitrate, avoid skin contact.Clean any spillages immediately. Wear protective clothing and gloves and dispose as chemical waste.

fluorescent-silver-staining-of-proteins-in-polyacrylamide-gels

Research

JoVE Journal

Biochemistry

Characterization of Multi-subunit Protein Complexes of Human MxA Using Non-denaturing Polyacrylamide Gel-electrophoresis

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral effector protein MxA exerts a broad antiviral activity against many viruses. MxA is a dynamin-like GTPase and has the capacity to form oligomeric structures of higher order. However, whether oligomerization of MxA is required for its antiviral activity is an issue of debate. We describe here a simple protocol to assess the oligomeric state of endogenously or ectopically expressed MxA in the cytoplasmic fraction of human cell lines by non-denaturing polyacrylamide gel electrophoresis (PAGE) in combination with Western blot analysis. A critical step of the protocol is the choice of detergents to prevent aggregation and/or precipitation of proteins particularly associated with cellular membranes such as MxA, without interfering with its enzymatic activity. Another crucial aspect of the protocol is the irreversible protection of the free thiol groups of cysteine residues by iodoacetamide to prevent artificial interactions of the protein. This protocol is suitable for a simple assessment of the oligomeric state of MxA and furthermore allows a direct correlation of the antiviral activity of MxA interface mutants with their respective oligomeric states.

This article describes a simple and rapid protocol to evaluate the oligomeric state of the dynamin-like GTPase MxA protein from lysates of human cells using a combination of non-denaturing PAGE with western blot analysis.

The formation of oligomeric complexes is a crucial prerequisite for the proper structure and function of many proteins. The interferon-induced antiviral ...

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where real-time measurements of fluorescence and currents simultaneously report on local rearrangements and global function, respectively1. While high-resolution structural techniques such as cryo-electron microscopy or X-ray crystallography provide static images of the proteins of interest, VCF provides dynamic structural data that allows us to link the structural rearrangements (fluorescence) to dynamic functional data (electrophysiology). Until recently, the thiol-reactive chemistry used for site-directed fluorescent labeling of the proteins restricted the scope of the approach because all accessible cysteines, including endogenous ones, will be labeled. It was thus required to construct proteins free of endogenous cysteines. Labeling was also restricted to sites accessible from the extracellular side. This changed with the use of Fluorescent Unnatural Amino Acids (fUAA) to specifically incorporate a small fluorescent probe in response to stop codon suppression using an orthogonal tRNA and tRNA synthetase pair2. The VCF improvement only requires a two-step injection procedure of DNA injection (tRNA/synthetase pair) followed by RNA/fUAA co-injection. Now, labelling both intracellular and buried sites is possible, and the use of VCF has expanded significantly. The VCF technique thereby becomes attractive for studying a wide range of proteins and &#8211; more importantly &#8211; allows investigating numerous cytosolic regulatory mechanisms.

Voltage-clamp Fluorometry in Xenopus Oocytes Using Fluorescent Unnatural Amino Acids

This article describes an enhancement of conventional Voltage-Clamp Fluorometry (VCF) where Fluorescent Unnatural Amino Acids (fUAA) are used instead of maleimide dyes, to probe structural rearrangements in ion channels. The procedure includes Xenopus oocyte DNA injection, RNA/fUAA coinjection, and simultaneous current and fluorescence measurements.

Voltage-Clamp Fluorometry (VCF) has been the technique of choice to investigate the structure and function of electrogenic membrane proteins where ...

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and podocytes with their slit diaphragms. The delicate structure of the glomerular filter, especially the slit diaphragm, relies on the interplay of diverse cell surface proteins. Studying these cell surface proteins has so far been limited to in vitro studies or histologic analysis. Here, we present a murine in vivo biotinylation labeling method, which enables the study of glomerular cell surface proteins under physiologic and pathophysiologic conditions. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of a protein of interest. Semi-quantitation of glomerular cell surface abundance is readily available with this novel method, and all proteins accessible to biotin perfusion and immunoprecipitation can be studied. In addition, isolation of glomeruli with or without biotinylation enables further analysis of glomerular RNA and protein as well as primary glomerular cell culture (i.e., primary podocyte cell culture).

Isolation of Glomeruli and In Vivo Labeling of Glomerular Cell Surface Proteins

Here we present a protocol for murine in vivo labeling of glomerular cell surface proteins with biotin. This protocol contains information on how to perfuse mouse kidneys, isolate glomeruli, and perform endogenous immunoprecipitation of the protein of interest.

Proteinuria results from the disruption of the glomerular filter that is composed of the fenestrated endothelium, glomerular basement membrane, and ...

How to Quantify the Fraction of Photoactivated Fluorescent Proteins in Bulk and in Live Cells

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells and protein ensembles. Thus far, no method has been available to quantify in bulk and in live cells how many of the PA-FPs expressed are photoactivated to fluoresce.
Here, we present a protocol involving internal rulers, i.e., genetically coupled spectrally distinct (photoactivatable) fluorescent proteins, to ratiometrically quantify the fraction of all PA-FPs expressed in a cell that are switched on to be fluorescent. Using this protocol, we show that different modes of photoactivation yielded different photoactivation efficiencies. Short high-power photoactivation with a confocal laser scanning microscope (CLSM) resulted in up to four times lower photoactivation efficiency than hundreds of low-level exposures applied by CLSM or a short pulse applied by widefield illumination. While the protocol has been exemplified here for (PA-)GFP and (PA-)Cherry, it can in principle be applied to any spectrally distinct photoactivatable or photoconvertible fluorescent protein pair and any experimental set-up.

Here, we present a protocol that involves genetically coupled spectrally distinct photoactivatable and fluorescent proteins. These fluorescent protein chimeras permit quantification of the PA-FP fraction that is photoactivated to be fluorescent, i.e., the photoactivation efficiency. The protocol reveals that different modes of photoactivation yield different photoactivation efficiencies.

Photoactivatable and -convertible fluorescent proteins (PA-FPs) have been used in fluorescence live-cell microscopy for analyzing the dynamics of cells ...

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

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, they are important drug targets. We have developed a magnetic-agarose-bead-based assay platform for the investigation of proteolytic activity, which is based on the use of recombinant fusion protein substrates. In order to demonstrate the use of this assay system, a protocol is presented on the example of human immunodeficiency virus type 1 (HIV-1) protease. The introduced assay platform can be utilized efficiently in the biochemical characterization of proteases, including enzyme activity measurements in mutagenesis, kinetic, inhibition, or specificity studies, and it may be suitable for high-throughput substrate screening or may be adapted to other proteolytic enzymes.
In this assay system, the applied substrates contain N-terminal hexahistidine (His6) and maltose-binding protein (MBP) tags, cleavage sites for tobacco etch virus (TEV) and HIV-1 proteases, and a C-terminal fluorescent protein. The substrates can be efficiently produced in Escherichia coli cells and easily purified using nickel (Ni)-chelate-coated beads. During the assay, the proteolytic cleavage of bead-attached substrates leads to the release of fluorescent cleavage fragments, which can be measured by fluorimetry. Additionally, cleavage reactions can be analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). A protocol for the in-gel renaturation of assay components is also described, as partial renaturation of fluorescent proteins enables their detection based on molecular weight and fluorescence.

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.

Proteases are intensively studied enzymes due to their essential roles in several biological pathways of living organisms and in pathogenesis; therefore, ...

Fluorescent Visualization of Mango-tagged RNA in Polyacrylamide Gels via a Poststaining Method

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, respectively. As many gel-imaging techniques use nonspecific stains or expensive fluorophore probes, sensitive, discriminating, and economical gel-imaging methodologies are highly desirable. RNA Mango core sequences are small (19&#8211;22 nt) sequence motifs that, when closed by an arbitrary RNA stem, can be simply and inexpensively appended to an RNA of interest. These Mango tags bind with high affinity and specificity to a thiazole-orange fluorophore ligand called TO1-Biotin, which becomes thousands of times more fluorescent upon binding. Here we show that Mango I, II, III, and IV can be used to specifically image RNA in gels with high sensitivity. As little as 62.5 fmol of RNA in native gels and 125 fmol of RNA in denaturing gels can be detected by soaking gels in an imaging buffer containing potassium and 20 nM TO1-Biotin for 30 min. We demonstrate the specificity of the Mango-tagged system by imaging a Mango-tagged 6S bacterial RNA in the context of a complex mixture of total bacterial RNA.

Here we present a sensitive, rapid, and discriminating post-gel staining method to image RNAs tagged with RNA Mango aptamers I, II, III, or IV, using either native or denaturing polyacrylamide gel electrophoresis (PAGE) gels. After running standard PAGE gels, Mango-tagged RNA can be easily stained with TO1-Biotin and then analyzed using commonly available fluorescence readers.

Native and denaturing polyacrylamide gels are routinely used to characterize ribonucleoprotein (RNP) complex mobility and to measure RNA size, ...

Localization of SUMO-modified Proteins Using Fluorescent Sumo-trapping Proteins

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. This method utilizes a recombinant modified SUMO-trapping protein fragment, kmUTAG, derived from the Ulp1 SUMO protease of the stress-tolerant budding yeast Kluyveromyces marxianus. We have adapted the properties of the kmUTAG for the purpose of studying sumoylation in a variety of model systems without the use of antibodies. For the study of SUMO, KmUTAG has several advantages when compared to antibody-based approaches. This stress-tolerant SUMO-trapping reagent is produced recombinantly, it recognizes native SUMO isoforms from many species, and unlike commercially available antibodies it shows reduced affinity for free, unconjugated SUMO. Representative results shown here include the localization of SUMO conjugates in mammalian tissue culture cells and nematode oocytes.

SUMO is an essential and highly conserved, small ubiquitin-like modifier protein. In this protocol we are describing the use of a stress-tolerant recombinant SUMO-trapping protein (kmUTAG) to visualize native, untagged SUMO conjugates and their localization in a variety of cell types.

Here we are presenting a novel method to study the sumoylation of proteins and their sub-cellular localization in mammalian cells and nematode oocytes. ...

Characterizing Cellular Proteins with In-cell Fast Photochemical Oxidation of Proteins

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. FPOP uses a 248 nm excimer laser to photolyze hydrogen peroxide producing hydroxyl radicals. These radicals oxidatively modify solvent exposed side chains of 19 of the 20 amino acids. Recently, this method has been used in live cells (IC-FPOP) to study protein interactions in their native environment. The study of proteins in cells accounts for intermolecular crowding and various protein interactions that are disrupted for in vitro studies. A custom single cell flow system was designed to reduce cell aggregation and clogging during IC-FPOP. This flow system focuses the cells past the excimer laser individually, thus ensuring consistent irradiation. By comparing the extent of oxidation produced from FPOP to the protein&#39;s solvent accessibility calculated from a crystal structure, IC-FPOP can accurately probe the solvent accessible side chains of proteins.

Here, we characterize protein structure and interaction sites in living cells using a protein footprinting technique termed in-cell fast photochemical oxidation of proteins (IC-FPOP).

Fast photochemical oxidation of proteins (FPOP) is a hydroxyl radical protein footprinting method used to characterize protein structure and interactions. ...

Transverse Sectioning of Mature Rice (Oryza sativa L.) Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm phenotyping can be used to classify genotypes based on SG morphotype using scanning electron microscopic (SEM) analysis. SGs can be visualized using SEM by slicing through the kernel (pericarp, aleurone layers, and endosperm) and exposing the organellar contents. Current methods require the rice kernel to be embedded in plastic resin and sectioned using a microtome or embedded in a truncated pipette tip and sectioned by hand using a razor blade. The former method requires specialized equipment and is time-consuming, while the latter introduces a new host of problems depending on rice genotype. Chalky rice varieties, particularly, pose a problem for this type of sectioning due to the friable nature of their endosperm tissue. Presented here is a technique for preparing translucent and chalky rice kernel sections for microscopy, requiring only pipette tips and a scalpel blade. Preparing the sections within the confines of a pipette tip prevents rice kernel endosperm from shattering (for translucent or &#8216;vitreous&#8217; phenotypes) and crumbling (for chalky phenotypes). Using this technique, endosperm cell patterning and the structure of intact SGs can be observed.

Transverse Sectioning of Mature Rice Oryza sativa L. Kernels for Scanning Electron Microscopy Imaging Using Pipette Tips as Immobilization Support

This protocol allows for the preparation of transverse sections of cereal seeds (e.g., rice) for the analysis of endosperm and starch granule morphology using scanning electron microscopy.

Starch granules (SGs) exhibit different morphologies depending on the plant species, especially in the endosperm of the Poaceae family. Endosperm ...

DetectSyn: A Rapid, Unbiased Fluorescent Method to Detect Changes in Synapse Density

Synapses are the site of communication between neurons. Neuronal circuit strength is related to synaptic density, and the breakdown of synapses is characteristic of disease states like major depressive disorder (MDD) and Alzheimer's disease. Traditional techniques to investigate synapse numbers include genetic expression of fluorescent markers (e.g., green fluorescent protein (GFP)), dyes that fill a neuron (e.g., carbocyanine dye, DiI), and immunofluorescent detection of spine markers (e.g., postsynaptic density 95 (PSD95)). A major caveat to these proxy techniques is that they only identify postsynaptic changes. Yet, a synapse is a connection between a presynaptic terminal and a postsynaptic spine. The gold standard for measuring synapse formation/elimination requires time-consuming electron microscopy or array tomography techniques. These techniques require specialized training and costly equipment. Further, only a limited number of neurons can be assessed and are used to represent changes to an entire brain region. DetectSyn is a rapid fluorescent technique that identifies changes to synapse formation or elimination due to a disease state or drug activity. DetectSyn utilizes a rapid proximity ligation assay to detect juxtaposed pre- and postsynaptic proteins and standard fluorescent microscopy, a technique readily available to most laboratories. Fluorescent detection of the resulting puncta allows for quick and unbiased analysis of experiments. DetectSyn provides more representative results than electron microscopy because larger areas can be analyzed than a limited number of fluorescent neurons. Moreover, DetectSyn works for in vitro cultured neurons and fixed tissue slices. Finally, a method is provided to analyze the data collected from this technique. Overall, DetectSyn offers a procedure for detecting relative changes in synapse density across treatments or disease states and is more accessible than traditional techniques.

DetectSyn is an unbiased, rapid fluorescent assay that measures changes in relative synapse (pre- and postsynaptic engagement) number across treatments or disease states. This technique utilizes a proximity ligation technique that can be used both in cultured neurons and fixed tissue.