We sincerely acknowledge the members of our new home at LPENS for their warm welcome, and in particular, the ABCDJ team for all their help and support. J.P. acknowledges financial support from the Foundation ARC (Grant PJA 20191209604), and C.S. acknowledges financial support from the Human Frontiers Science Program Organization (Grant RGP0026/2020).

1. Preparation of buffers
NOTE: Use ultrapure H2O for all buffers. It does not have to be sterile. Filter all the solutions described in steps 1.1-1.4 with a 0.2 &#181;m syringe filter, aliquot in portions of 500 &#181;L-2 mL per tube depending on usage, and store at -20 &#176;C.
<ol>
	<li>Prepare 10% BSA by weighing 2 g of BSA into a 50 mL conical tube and filling to the 20 mL mark with H2O. Mix until the BSA is dissolved (about 30 min), and then make up the volume to 20 mL. 
	NOTE: Use high quality BSA (see Table of Materials). The 10% BSA solution is used in both bead preparation and the motility mix.</li>
	<li>For bead preparation, prepare Xb buffer (10 mM HEPES, 0.1 M KCl, 1 mM MgCl2, and 0.1 mM CaCl2, pH 7.5) as a 10x solution, and dilute before use (100 &#181;L of 10x Xb stock solution + 900 &#181;L of H2O). Prepare Xb/1% BSA by mixing 100 &#181;L of 10x Xb stock solution + 100 &#181;L of 10% BSA + 800 &#181;L of H2O.</li>
	<li>Prepare G-buffer (2 mM Tris, 0.2 mM CaCl2, 0.2 mM DTT, 2 mM ATP), which is the buffer used to dilute monomeric actin (G-actin). Adjust to pH 7, not pH 8 as traditionally used (see discussion).</li>
	<li>Prepare the motility buffer MB13 (10 mM HEPES, 1.5 mM ATP, 3 mM DTT, 1.5 mM MgCl2, 1 mM EGTA, 50 mM KCl, 1% BSA, pH 7.5). For some applications, 10x MB13 is useful. However, prepare 10x MB13 without BSA, as it leads to problems during pH adjustment. Add BSA from 10% stock solution (prepared in step 1.1) when reconstituting 1x MB13 from 10x MB13.</li>
</ol>
2. Preparation of protein solutions
NOTE: Use ultrapure H2O for all resuspensions. It does not have to be sterile. Handle all proteins on ice and aliquot into pre-chilled tubes. Manipulate gently so as not to produce bubbles, and never vortex protein solutions. For stocks to be stored at -80&#176;C, flash freezing in liquid nitrogen is not necessary. Adapt aliquot size to avoid more than about five freeze-thaw cycles, as this does not appear to affect the activity of any of the proteins. Working aliquots can be stored at -20 &#176;C for a few weeks.
<ol>
	<li>Prepare G-actin (rabbit skeletal muscle) solution as described below.
	<ol>
		<li>Pulse centrifuge actin powder (see Table of Materials) at 4 &#176;C to gather the solid at the bottom of the tube.</li>
		<li>Add H2O as per manufacturers&#39; instructions (1 mg protein in 100 &#181;L of cold H2O for unlabeled actin, 100 &#181;g protein in 100 &#181;L of cold H2O for ATTO-labeled actin).</li>
		<li>Let it sit on ice for at least 15 min. Mix gently by pipetting up and down, let it sit on ice at least another 15 min, and mix again. Pulse centrifuge at 4 &#176;C to collect the solution at the bottom of the tube, and remix.</li>
		<li>Prepare 10-50 &#181;L aliquots of unlabeled actin depending on use, and 20 &#181;L aliquots of ATTO-labeled actin. Store the aliquots at -80 &#176;C.</li>
		<li>To depolymerize actin oligomers that form during lyophilization and freezing, dilute an aliquot of resuspended actin ~8-fold in G-buffer spiked with additional ATP and DTT (for example, to 20&#181;L of resuspended actin solution from step 2.1.4, add 134 &#181;L of G-buffer, 0.32 &#181;L of 0.2 mM ATP and 0.16 &#181;L of 1 M DTT). For fluorescent labeling, add approximately 10% labeled actin; for example, add 5 &#181;L of ATTO-labeled actin to 40 &#181;L of diluted unlabeled actin. Let it depolymerize on ice with occasional mixing (pipetting) at least a few days to a week before measuring protein concentration by Bradford assay as described in step 3. 
		NOTE: Keep diluted unlabeled and fluorescent actin on ice in a cold room or refrigerator; never freeze or allow to warm. The preparation will continue depolymerizing over time, and when handled properly can be used for at least 6 months.</li>
	</ol>
	</li>
	<li>Resuspend Arp2/3 complex (porcine brain) (see Table of Materials) following the manufacturer&#39;s instructions (20 &#181;g protein in 20 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as described for actin in step 2.1. Combine the protein solutions from resuspending two tubes of powder to have a larger stock for reproducible experiments. Prepare 2 &#181;L aliquots and store at -80 &#176;C.</li>
	<li>Resuspend profilin (human recombinant) (see Table of Materials) at a 4x higher concentration than stipulated in the manufacturer&#39;s instructions (100 &#181;g protein in 25 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as for actin in step 2.1. Combine the protein solutions from resuspending two tubes of powder before determining protein concentration to have a larger stock for reproducible experiments. 
	NOTE: Keep on ice in a cold room or refrigerator; never freeze or allow to warm. When handled properly, resuspended profilin is good for at least 6 months to 1 year.</li>
	<li>Resuspend capping protein (&#945;1&#946;2, human recombinant) (see Table of Materials) following the manufacturer&#39;s instructions (50 &#181;g protein in 50 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing etc. on ice as described for actin in step 2.1. Chill 50 &#181;L of glycerol on ice and add the 50 &#181;L of resuspended capping protein to it; mix gently. Store at -20 &#176;C. 
	NOTE: The solution does not freeze and the activity is robust, so the solution can be kept as a single aliquot for months, or even years, if handled carefully. Mouse recombinant capping protein, the one most commonly used in the past in in vitro experiments13, will soon be commercially available.</li>
	<li>Resuspend gelsolin (human recombinant, His-tagged) (see Table of Materials) following the manufacturer&#39;s instructions (20 &#181;g protein in 20 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as described for actin in step 2.1. About 2 &#181;L of gelsolin is used per day of experiments; therefore, prepare large aliquots (5-10 &#181;L) and store at -80 &#176;C. 
	NOTE: The protocol with gelsolin is provided as an alternative. The use of capping protein in the place of gelsolin is recommended, either purchased or purified as in13.</li>
	<li>Resuspend VCA (human WASP-VCA, GST-tagged) (see Table of Materials) at 2x higher concentration than stipulated in the manufacturer&#39;s instructions (500 &#181;g protein in 250 &#181;L cold H2O), with the sequence of pulse centrifuging, mixing, etc. on ice as for actin in step 2.1. Make 10 &#181;L aliquots and store at -80 &#176;C. 
	&#8203;NOTE: Once SpVCA (human pVCA, streptavidin and His-tagged) is commercialized or if protein purification14 is possible, the use of SpVCA instead of VCA is recommended. VCA does not reproducibly give comets under the conditions described here.</li>
</ol>
3. Measurement of protein concentrations
<ol>
	<li>Construct a Bradford standard curve made of two overlapping serial dilutions of BSA. 
	NOTE: The standard curve only needs to be constructed every couple of months (or even less frequently) as long as the spectrophotometer does not change.
	<ol>
		<li>In row 1 of a microtube rack, place tubes for BSA dilution series #1: four 2 mL tubes followed by four 1.5 mL tubes. In row 3 of the rack, place tubes for BSA dilution series #2 as for series #1. In row 5 of the rack, place one 2 mL tube for each sample to be measured along with two 1.5 mL tubes. Add a single 1.5 mL tube in row 5 for the blank.</li>
		<li>Measure out Bradford Reagent (see Table of Materials) into the 1.5 mL tubes.
		<ol>
			<li>Fill a 15 mL conical tube to the top with Bradford Reagent for easier pipetting (excess will be returned to the stock bottle in the refrigerator) on ice. Take up 200 &#181;L Bradford Reagent and eject it back into the conical tube to wet the pipette tip. As the solution is viscous, pipette slowly to allow the solution to enter and leave the tip completely without making bubbles.</li>
			<li>With the &#34;pre-wet&#34; tip, slowly pipette 200 &#181;L of Bradford Reagent into each of the 1.5 mL tubes in the rack (four for each BSA dilution, one for the blank and two for each sample to be measured). Do this first to allow the Bradford Reagent to thoroughly warm to room temperature before mixing with protein solutions. Return the remaining contents of the 15 mL conical tube to the bottle.</li>
		</ol>
		</li>
		<li>Measure out H2O into the 2 mL tubes. In row 1 of the rack, add 1,990 &#181;L H2O to the first tube, and 900 &#181;L to the three other tubes. For row 3, add 1,992.5 &#181;L to the first tube and 900 &#181;L to the three other tubes. Add 2,000 &#181;L H2O to each of the sample tubes in row 5. 
		NOTE: For all volumes larger than 1,000 &#181;L, use a 1,000 &#181;L pipette, but administer the full amount by pipetting twice. It is important to get everything ready before starting the subsequent steps, in order to avoid leaving proteins highly diluted in H2O for long periods of time.</li>
		<li>To prepare BSA dilution series #1, mix 10 &#181;L of calibrated 2 mg/mL BSA (see Table of Materials) into the tube with 1,990 &#181;L H2O to make a 10 &#181;g/mL solution. From this, make three serial dilutions (5 &#181;g/mL, 2.5 &#181;g/mL, and 1.25 &#181;g/mL BSA) by transferring 900 &#181;L of each solution into the next tube (containing 900 &#181;L H2O).</li>
		<li>To prepare BSA dilution series #2, mix 7.5 &#181;L of calibrated 2 mg/mL BSA into the tube with 1,992.5 &#181;L H2O to make a 7.5 &#181;g/mL solution. From this, make three serial dilutions (3.75 &#181;g/mL, 1.875 &#181;g/mL and 0.9375 &#181;g/mL BSA) by transferring 900 &#181;L of each solution into the next tube (containing 900 &#181;L H2O).</li>
		<li>Mix and read absorbance to generate the standard curve. Add 800 &#181;L H2O to the Bradford reagent tube for the blank, and start the timer. As efficiently as possible without making bubbles, mix 800 &#181;L of each BSA standard with 200 &#181;L Bradford reagent in the prepared tubes. Once all the standards have been mixed with Bradford reagent (&#60; 5 min), pour each standard into a disposable cuvette and read the absorbance at 600 nm in the spectrophotometer after blanking the machine. 
		NOTE: The same cuvette can be used to read the whole series if the least concentrated standard is read first and the cuvette is well-emptied between reads. Redo the standard curve until the linear fit has an R value of at least 0.99. Only after pipetting and gentle mixing is mastered, proceed to reading samples.</li>
	</ol>
	</li>
	<li>Measure concentrations of actin and actin-binding proteins
	<ol>
		<li>In the 2 mL tubes containing 2,000 &#181;L of H2O (prepared in step 3.1.3), gently mix in the following: 2 &#181;L each of Arp2/3 complex and profilin, 4 &#181;L of labeled G-actin, 5 &#181;L each of gelsolin and VCA and 8 &#181;L of capping protein (human recombinant). Immediately take 800 &#181;L of the solution and mix with the already prepared Bradford reagent, and then repeat to have two readings within 5%-10 % of each other for each sample. A larger difference indicates a problem with resuspension or handling. Read within a few minutes of mixing with Bradford reagent. 
		NOTE: If using a standard curve from another day, only the blank from step 3.1.6 has to be prepared, in addition to the samples.</li>
	</ol>
	</li>
	<li>Calculate the concentrations of actin and actin-binding proteins using the standard curve and the dilution factor. Redo the measurement for each new resuspension. Molecular weights to convert mg/mL readings obtained via Bradford assay to &#181;M are: actin 43 kD, Arp2/3 complex 224 kD, profilin 15 kD, gelsolin 95 kD, capping protein 68 kD (human recombinant) or 63.5 kD (mouse recombinant), VCA 43 kD, and SpVCA 54 kD (monomer molecular weight).</li>
</ol>
4. Coating of beads
<ol>
	<li>Pre-chill the centrifuge to 4 &#176;C and set the agitating dry block (see Table of Materials) to 18 &#176;C.</li>
	<li>Wash the beads: pipette 50 &#181;L of Xb buffer into a 1.5 mL microcentrifuge tube, add 9 &#181;L of 4.5 &#181;m diameter bead suspension or 2 &#181;L of 1 &#181;m diameter bead suspension (2.5 % w/v suspension) (see Table of Materials). Mix thoroughly and centrifuge the samples at 20,000 x g for 10 min at 4 &#176;C. 
	NOTE: Total bead surface area of both bead sizes is 3 cm2: 
	<img alt="Equation 1" src="/files/ftp_upload/64261/64261eq01.jpg" style="margin: auto;" /> 
	derived by calculating the number of beads and then their total surface using classic equations for the volume and surface area of spheres. Other sizes of beads can be used if amounts are adjusted to keep the total surface area constant at 3 cm2.</li>
	<li>Coat the beads: carefully remove the supernatant without disturbing the beads, and resuspend the bead pellet in 40 &#181;L of 2 &#181;M SpVCA (or 7 &#181;M VCA) in Xb buffer by gentle pipetting. Agitate at 18 &#176;C, 1,000 rpm for 20 min.</li>
	<li>Wash coated beads: centrifuge the mix (20,000 x g for 10 min at 4 &#176;C) and carefully remove the supernatant. Resuspend the beads in 50 &#181;L of cold Xb/1% BSA, and centrifuge at 20,000 x g for 10 min at 4 &#176;C. Remove the supernatant and repeat the washing step 1x.</li>
	<li>Resuspend the coated bead pellet from step 4.4 in 120 &#181;L cold Xb/1% BSA for both sizes of beads so that the amount of bead surface area/&#181;L of bead solution is the same. Store on ice in a refrigerator or cold room. Coated beads will continue to function normally for at least several weeks.</li>
</ol>
5. Preparing the motility mix and slides for observation
NOTE: The total volume of the motility mix is 8.4 &#181;L to allow for about 25 &#181;m clearance between the slide and the 18 mm x 18 mm coverslip so beads of all sizes (up to 10 &#181;m diameter) are not squeezed. The basic motility mix is approximately 5 &#181;M G-actin (10% labeled fluorescent actin) with 5 &#181;M profilin, 50 nM Arp2/3 complex, and 25 nM capping protein (or 240 nM gelsolin).
<ol>
	<li>Prepare the motility reaction mixture. Exact amounts of actin (and therefore profilin) depend on the concentration calculated in step 3.3, but a representative reaction is as follows. Mix on ice, in this order: 3.2 &#181;L of MB13, 1.5 &#181;L of profilin at 30 &#181;M diluted in MB13, 1 &#181;L of capping protein at 0.21 &#181;M diluted in MB13 or gelsolin diluted to 2 &#181;M in MB13, 1 &#181;L of Arp2/3 complex at 0.47 &#181;M diluted in MB13, 0.2 &#181;L of bead suspension (vortex just before use), and 1.5 &#181;L of actin at 30 &#181;M in G-buffer. Mix well but quickly and start the timer.</li>
	<li>Spot the entire motility reaction mixture on a slide. Cover with an 18 mm x 18 mm coverslip and seal the coverslip with melted VALAP using a small paintbrush. VALAP is a mix of lanolin, paraffin, and petroleum jelly (see Table of Materials) 1:1:1 by weight, melted and stirred together.</li>
</ol>
6. Microscopy observation
<ol>
	<li>Observe motility reactions immediately using a 100x objective on either an upright or an inverted microscope (see Table of Materials), equipped with phase contrast and/or epifluorescence microscopy (GFP cube, see Table of Materials). Observations are done at room temperature (23-25 &#176;C).
	<ol>
		<li>To obtain average displacement speeds for a whole population of beads, record phase contrast or fluorescent still images over time by scanning the entire slide. Measure comet length by hand and plot versus time. The slope of the linear fit is the average growth speed.</li>
		<li>To evaluate the speed of individual beads, collect time-lapse movies in phase contrast microscopy. Depending on the bead speed and the resolution required, take frames every 1-10 s. Use the tracking tool of any image processing program to obtain bead speeds and trajectories.</li>
	</ol>
	</li>
</ol>

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M., Staiger, C. J., Min, J. -. K., Simonetti, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+gelsolin+family+of+actin+regulatory+proteins:+modular+structures,+versatile+functions.">The gelsolin family of actin regulatory proteins: modular structures, versatile functions.</a> FEBS Letters. 552 (2-3), 75-81 (2003).</li><li>Abou-Ghali, 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=Capping+protein+is+not+necessary+for+polarized+actin+network+growth+and+actin+based+motility.">Capping protein is not necessary for polarized actin network growth and actin based motility.</a> Journal of Biological Chemistry. 295, 15366-15375 (2020).</li><li>Yarar, D., D'Alessio, J. A., Jeng, R. L., Welch, M. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Motility+determinants+in+WASP+family+proteins.">Motility determinants in WASP family proteins.</a> Molecular Biology of the Cell. 13 (11), 4045-4059 (2002).</li><li>Padrick, S. B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hierarchical+regulation+of+WASP/WAVE+proteins.">Hierarchical regulation of WASP/WAVE proteins.</a> Molecular Cell. 32 (3), 426-438 (2008).</li><li>Bussonier, 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=Mechanical+detection+of+a+long-range+actin+network+emanating+from+a+biomimetic+cortex.">Mechanical detection of a long-range actin network emanating from a biomimetic cortex.</a> Biophysical Journal. 107 (4), 854-862 (2014).</li><li>Paluch, E., vander Gucht, J., Joanny, J. -. F., Sykes, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deformations+in+actin+comets+from+rocketing+beads.">Deformations in actin comets from rocketing beads.</a> Biophysical Journal. 91 (8), 3113-3122 (2006).</li></ol>

The authors declare that they have no conflicts of interest with the contents of this article.

The protocol detailed here describes how to obtain actin network growth on bead surfaces, comet formation, and bead motility using commercially available proteins. However, sometimes comets are not reproducibly observed or are inhomogeneous between the slide and coverslip. The following discussion emphasizes some key points in the protocol and suggests some parameters that can be adjusted. One factor to keep in mind is that comet formation and bead speed are affected by temperature, with temperatures much above 25 &#176;C or much below 23 &#176;C negatively impacting comet formation and giving irreproducible data. Use of a temperature-controlled microscope or a microscope in a climate-controlled room is strongly recommended. Although fluorescently labeled actin is often included in the motility mix to observe comets by fluorescence microscopy, once comets are more than a bead diameter in length, they are also visible by phase contrast microscopy as a dark smear next to the bead. Phase contrast visualization is more appropriate for time-lapse imaging as some phototoxicity is associated with fluorescence imaging even via spinning disc. Because beads settle over time, an inverted microscope produces less horizontal bead drift than an upright one and is more appropriate for movies. The use of molten VALAP to seal slides is important as substances such as nail polish interfere with comet formation. Large quantities of VALAP can be made in a beaker, and then scooped out to refill smaller beakers more amenable to rapid melting. VALAP is good for years at room temperature.
Another key technical aspect is meticulous buffer and motility mix preparation. Care should be taken when preparing MB13, in particular at the pH adjustment step. The pH of MB13 should be adjusted rapidly to neutral with NaOH to avoid ATP hydrolysis, but not too quickly as the EGTA solubilizes as the pH approaches neutral. EGTA is a key ingredient because it complexes the calcium bound to actin, giving in the motility mix the more active magnesium form16. MB13 prepared too quickly or too slowly gives suboptimal comet formation or even none at all. An additional key point is to keep careful track of KCl concentration in the motility mix when playing with conditions. For example, when using 1x MB13 in the reaction mix and diluting profilin, capping protein, and the Arp2/3 complex in MB13, the final KCl concentration in the motility reaction is about 40-50 mM due to dilution by G-buffer. This concentration gives the best results in the comet assay, and any more than 60 mM KCl decreases Arp2/3 complex nucleating activity.
On the protein side of things, a critical technical aspect of obtaining actin comets is proper handling of commercial actin-binding proteins, in particular precise pipetting of microliter quantities. The linearity of the Bradford standard curve is a good test of pipetting and the curve can then be used for routine measurements of protein concentrations. Indeed, when using resuspended commercial proteins for the comet procedure, it is important to always verify protein concentrations, as batch variability and user error during resuspension can lead to differences between real and expected concentrations. Sometimes small differences in protein concentrations can lead to the complete absence of comets.
Another important aspect of the method presented here is the use of profilin-complexed G-actin as the fuel for polymerization. Historically, in vitro systems used pre-polymerized filamentous actin (F-actin) as the actin source: depolymerization in the bulk fed polymerization on the surface10,17. This had the advantage of controlling G-actin levels, but added a layer of complexity requiring additional components to catalyze depolymerization. Since turnover of the actin network is not necessary for force production and motility, which are fueled by nucleation and polymerization at the surface of the bead, while actin depolymerization factors such as ADF/cofilin act on the aged networks far from the surface18, most in vitro reconstitution of actin-based motility is now done without turnover for simplicity. However, there are some drawbacks to using G-actin. First, when using commercial actin, which has been lyophilized, oligomers are present. The depolymerization steps described here are very important in obtaining reproducible results. In particular, although G-buffer is traditionally adjusted to pH 8, lower pH (pH 7, for example) appears to work better in the assays described in this article, possibly because low pH enhances depolymerization19. Another disadvantage of using G-actin is that once placed in salt conditions permissive to polymerization, spontaneous nucleation occurs and F-actin forms in the bulk as well as on the bead surface. Complexing G-actin with profilin suppresses spontaneous nucleation in the bulk and pointed end polymerization, thereby focusing both nucleation and barbed end polymerization at the surface20. Profilin-G-actin is physiologically relevant, as much of the actin in the cell is present in this form21. Here, a 1:1 ratio of profilin:actin is used; however, higher ratios (for example 3:1) more thoroughly inhibit polymerization in the bulk, although higher ratios also inhibit the Arp2/3 complex and barbed end elongation to some extent22,23.
Capping activity is also key for comet formation since it ensures insertion of new actin at the surface via cycles of nucleation by surface-activated Arp2/3 complex24,25. Without capping, actin clouds do not break symmetry to form comets because polymerization at the surface does not build up enough tension to break open the cloud26. In the past, we have used home-purified recombinant mouse capping protein13, but tests performed for this article indicate that commercially available recombinant human capping protein is equally effective, as is commercially available gelsolin, although 10x more gelsolin has to be used, and for certain applications, it may not be appropriate as it has actin severing activity as well as capping27.
Finally the robustness of this method resides in the use of a very active Arp2/3 complex activator, streptavidin-pVCA (SpVCA)28. SpVCA includes the profilin-G-actin binding domain of WASP (the p domain) in addition to the Arp2/3 complex binding domain as this is found to be most efficient in profilin-G-actin conditions29. More importantly, the use of the streptavidin tag, originally introduced to allow surface functionalization via the biotin-streptavidin link, has the additional effect of increasing Arp2/3 complex activation, presumably due to the fact that streptavidin is a tetramer and thus clusters the activator, known to increase Arp2/3 complex activity30. Commercially-produced SpVCA is currently in development and will soon be available for purchase. It should further be noted that, although 40 &#181;L of 2 &#181;M SpVCA is routinely used to coat 3 cm2 of bead surface, other coating concentrations (higher and lower) also work, and playing with these conditions gives different comet growth speeds and morphologies. Indeed, when comets do not form or comet size is not homogenous on the slide, different coating conditions should be tested, as well as different KCl and profilin concentrations in the motility mix. The concentrations of actin, Arp2/3 complex, and capping protein in the motility mix can also be altered to optimize comet formation, but in our hands, changing these proportions often gives confusing results.
To conclude, the methods described here produce actin assembly on bead surfaces and motility, but any surface that can be functionalized with SpVCA can be used. In cases where adsorption as described here does not work, the streptavidin moiety can be used to attach SpVCA to the surface of interest after biotinylation. The actin structures thus formed, comets or otherwise, can be used for testing different biochemical and biophysical aspects of actin networks, and are especially appropriate for physical manipulations with micropipettes, optical tweezers, and laser ablations15,26,31,32. In addition to its uses to the research community, the approach described here is appropriate as a teaching tool for undergraduate biophysics students to study active matter concepts such as symmetry breaking and self-organization.

Actin polymerization in cells is spatially and temporally controlled by tight regulation of actin filament nucleation downstream of cell signaling1. Nucleation occurs via the formation of an actin trimer, and then both ends of the nascent filament polymerize spontaneously, although one end is more dynamic (the barbed end) than the other (the pointed end)2. When nucleation and barbed end polymerization are directed toward a surface, they produce enough force (in the pico-to-nano Newton range) to push out the cell membrane for movement and to move micron-sized objects inside the cell with ATP hydrolysis as the energy source3. Some examples include Listeria monocytogenes bacteria that use actin comets to spread from cell to cell, and mitochondria, where actin comet-based movement is important for randomized inheritance during mitosis4,5. Actin comets on endosomes and other intracellular vesicles are implicated in detachment from donor membranes6,7,8.
With the method presented here, the signaling aspects of cellular actin polymerization are bypassed, and actin polymerization is produced on micrometric polystyrene beads by coating them with activators of branched actin nucleation, specifically the active domain of the human WASP protein, VCA (also called WA or WCA)1. The coated beads are then incubated in a mix containing the ingredients necessary for actin polymerization, including the main actin polymerization nucleator in cells, the Arp2/3 complex, which is activated by VCA at the bead surface to form new filaments as branches off the sides of daughter filaments1. Actin initially polymerizes uniformly around the bead, but then spontaneously breaks symmetry to create an actin comet that pushes the bead forward, thereby recreating cell-like protrusive networks and comets in a controlled manner. Similar approaches with beads and other coated surfaces have been used in the past by us and others to study the biochemistry and biophysics of actin polymerization9,10,11,12, but extensive expertise in actin-binding proteins was required for these experiments. The protocol presented here describes how to robustly create actin comets and motility entirely with commercially available (or soon to be available) reagents, making this approach accessible to anyone, including in an educational setting for teaching biophysical concepts. Key features include the importance of gentle and reliable pipetting, the use of profilin-complexed monomer as the actin source, and the essentiality of using a highly active Apr2/3 complex activator as a bead coating reagent.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>Actin, rabbit muscle, Alexa Fluor 488 conjugate&#160;</td>
			<td>Invitrogen (ThermoFisher Scientific)</td>
			<td>A12373 (recently discontinued)</td>
			<td>This product can be replaced with ATTO-488 actin from Hypermol.</td>
		</tr>
		<tr>
			<td>Actin, rabbit muscle, ATTO-488</td>
			<td>Hypermol</td>
			<td>8153</td>
			<td></td>
		</tr>
		<tr>
			<td>Actin, rabbit skeletal muscle</td>
			<td>Cytoskeleton</td>
			<td>AKL99</td>
			<td></td>
		</tr>
		<tr>
			<td>Arp2/3 complex</td>
			<td>Cytoskeleton</td>
			<td>RP01P</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>Sigma</td>
			<td>A7699</td>
			<td></td>
		</tr>
		<tr>
			<td>BioSpectrometer, basic</td>
			<td>Eppendorf</td>
			<td>035739</td>
			<td></td>
		</tr>
		<tr>
			<td>Bradford Reagent</td>
			<td>Bio-Rad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA, high quality</td>
			<td>Sigma</td>
			<td>A3059</td>
			<td></td>
		</tr>
		<tr>
			<td>BSA standard 2 mg/mL (Pierce)</td>
			<td>Thermo Scientific</td>
			<td>23209</td>
			<td></td>
		</tr>
		<tr>
			<td>Capping protein (a1b2, mouse recombinant)</td>
			<td>Home-purified (Reference 13)</td>
			<td></td>
			<td>This product will soon be commercially available from Cytoskeleton.</td>
		</tr>
		<tr>
			<td>Capping protein (a1b2, human recombinant)</td>
			<td>Hypermol</td>
			<td>8322</td>
			<td></td>
		</tr>
		<tr>
			<td>Cube, GFP: U-MNIBA3 or U-MWB2</td>
			<td>Olympus</td>
			<td>discontinued</td>
			<td>Any GFP cube, adapted to the microscope being used, can be used.</td>
		</tr>
		<tr>
			<td>Dry block, agitating: ThermoMixer C (refrigerated)</td>
			<td>Eppendorf</td>
			<td>035963</td>
			<td></td>
		</tr>
		<tr>
			<td>** with SmartBlock, 24 microtubes 2 mL</td>
			<td>Eppendorf</td>
			<td>035969</td>
			<td></td>
		</tr>
		<tr>
			<td>Gelsolin (human recombinant, His-tagged)</td>
			<td>Cytoskeleton</td>
			<td>HPG6</td>
			<td></td>
		</tr>
		<tr>
			<td>Lanolin</td>
			<td>Sigma</td>
			<td>49909</td>
			<td></td>
		</tr>
		<tr>
			<td>Microcentrifuge 5427R + rotor</td>
			<td>Eppendorf</td>
			<td>934126</td>
			<td></td>
		</tr>
		<tr>
			<td>Microscope, upright: BX51</td>
			<td>Olympus</td>
			<td>discontinued</td>
			<td>Any epifluorescence upright microscope equipped with phase contrast optics can be used.</td>
		</tr>
		<tr>
			<td>Microscope, inverted: IX70</td>
			<td>Olympus</td>
			<td>discontinued</td>
			<td>Any epifluorescence inverted microscope equipped with phase contrast optics can be used.</td>
		</tr>
		<tr>
			<td>Paraffin</td>
			<td>Sigma</td>
			<td>76244</td>
			<td></td>
		</tr>
		<tr>
			<td>Petroleum jelly: Vaseline</td>
			<td>Sigma</td>
			<td>16415</td>
			<td></td>
		</tr>
		<tr>
			<td>Pipettes Research Plus</td>
			<td>Eppendorf</td>
			<td></td>
			<td>Gilson pipettes don&#39;t work as well for delivery of very small volumes (0.5 &#181;L for example).</td>
		</tr>
		<tr>
			<td>**10 &#181;L</td>
			<td></td>
			<td>933954</td>
			<td></td>
		</tr>
		<tr>
			<td>**2.5 &#181;L</td>
			<td></td>
			<td>933953</td>
			<td>These two sizes are essential, but the use of high-quality pipettes (a full Research Plus set for example) is recommended.</td>
		</tr>
		<tr>
			<td>Polystyrene carboxylate beads</td>
			<td>Polysciences</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>**approx. 1 &#181;m diameter</td>
			<td></td>
			<td>08226</td>
			<td></td>
		</tr>
		<tr>
			<td>**approx. 4.5 &#181;m diameter</td>
			<td></td>
			<td>17140-5</td>
			<td></td>
		</tr>
		<tr>
			<td>Profilin 1 (human recombinant, untagged)</td>
			<td>Cytoskeleton</td>
			<td>PR02</td>
			<td></td>
		</tr>
		<tr>
			<td>SpVCA (human WASP pVCA domain, N-ter His-tag, C-ter Streptavidin tag)</td>
			<td>Home-purified (Reference 14)</td>
			<td></td>
			<td>This product will soon be commercially available from Cytoskeleton.</td>
		</tr>
		<tr>
			<td>VCA (human WASP VCA domain, GST-tagged)</td>
			<td>Cytoskeleton</td>
			<td>VCG03</td>
			<td></td>
		</tr>
	</tbody>
</table>

reconstitution of actin-based motility with commercially available proteins

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a dynamic network at the surface of the cell, organelle, or bacterium. The biochemical and mechanical basis of force production during this process can be studied by reproducing actin-based movement in an acellular manner on inert surfaces such as beads that are functionalized and incubated with a controlled set of components. Under the appropriate conditions, an elastic actin network assembles at the bead surface and breaks open due to the stress generated by network growth, forming an "actin comet" that propels the bead forward. However, such experiments require the purification of a host of different actin-binding proteins, often putting them beyond the reach of non-specialists. This article details a protocol for reproducibly obtaining actin comets and motility of beads using commercially available reagents. Bead coating, bead size, and motility mixture can be altered to observe the effect on bead speed, trajectories, and other parameters. This assay can be used for testing the biochemical activities of different actin-binding proteins, and for performing quantitative physical measurements that shed light on active matter properties of actin networks. This will be a useful tool for the community, enabling the study of in vitro actin-based motility without expert knowledge in actin-binding protein purification.

One of the key aspects of reproducibly creating actin comets on beads is gentle and precise pipetting of delicate actin-binding proteins. Generating a Bradford standard curve is a good way of evaluating pipetting skills. Figure 1A,B shows the tubes for the standard curve and an example of what the two serial dilutions of BSA look like once mixed with Bradford reagent. Note the graded blue hue (higher protein concentration gives a bluer solution). When read in the spectrophotometer and plotted, these solutions give a standard curve as shown in Figure 1C. To practice careful pipetting, the assay should be repeated until the linear correlation factor is 0.999, as shown.
Once the concentrations of commercial resuspended proteins have been carefully evaluated via the Bradford assay, coated beads and motility mix are prepared and mixed together. Figure 2A shows representative images of the different stages of comet formation: actin clouds form within minutes of mixing SpVCA-coated beads and motility medium; cloud polarization occurs at ~5 min and comet production at 15-20 min. The actin comets, visible with both epifluorescence and phase contrast microscopy (Figure 2A), continue to elongate for many hours, but a consistent speed is not maintained so bead motility is normally evaluated within 1 h. On the other hand, it takes 30 min with VCA-coated beads to obtain bright actin clouds (Figure 2B), and no comets form, although symmetry starts to break at 1-2 h (arrow in Figure 2B) and clouds show polarization after overnight incubation.
Figure 3 shows an example of bead velocity evaluation in the presence of capping protein. Since all beads break symmetry at approximately the same time, &#34;pseudo-time-lapse&#34; recordings are performed where the slide is scanned and pictures of the whole population of comets are taken over time (Figure 3A). Comets do not depolymerize; therefore, the increase in comet lengths measured over time can be used to calculate displacement speed (Figure 3B). Gelsolin can be used in the place of capping protein for comet formation if 10x more gelsolin is added to compensate for its reduced capping activity. Comets formed in the presence of gelsolin are qualitatively the same and move at approximately the same speeds as beads with capping protein (Figure 3C). Capping activity is key for concentrating polymerization at the surface of the bead, and when neither capping protein nor gelsolin are included in the motility mix, actin clouds never polarize to form comets, although bright actin clouds form around the beads (Figure 3D). Comets on beads can be used to measure actin-based force production in different biochemical contexts by altering the motility mix and observing the result on motility using different micromanipulation techniques, for example15.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/64261/64261fig01.jpg" /> 
Figure 1: Bradford standard curve. (A) Picture of how to set up the tubes for making the Bradford standard curve. Sample tubes are not shown. (B) Picture of the two overlapping BSA serial dilutions once mixed with Bradford reagent. (C) The absorbances at 600 nm of the solutions shown in (B) are measured in the spectrophotometer and are plotted as a function of the protein concentrations of the BSA solutions. The linear fit is used to calculate the sample concentration. The correlation factor, R, of the linear fit is 0.999. <a href="https://www.jove.com/files/ftp_upload/64261/64261fig01large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 2" class="xfigimg" src="/files/ftp_upload/64261/64261fig02.jpg" /> 
Figure 2: Comet formation on SpVCA-coated beads as opposed to VCA-coated beads. (A) Representative SpVCA-coated beads (different bead in each image) shown over time. Time from the moment of mixing is indicated. Actin clouds form immediately, and cloud polarization gives comets, which continue to elongate for hours. (B) Representative VCA-coated beads (different bead in each image) shown over time. More than 1 h is necessary to see the beginnings of actin cloud polarization (arrow) and even long incubations do not produce comets. All images are of 4.5 &#181;m diameter beads, epifluorescence imaging of fluorescent actin paired with phase contrast visualization for the 15 and 20 min time-points in (A), scale bars = 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64261/64261fig02large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" class="xfigimg" src="/files/ftp_upload/64261/64261fig03.jpg" /> 
Figure 3: Comets and bead velocity analysis. (A) and (C) In the presence of either capping protein (CP) or gelsolin, actin clouds polarize to form comets in the first 20 min of reaction, and comets elongate over time. Time from mixing indicated for each image; each image is a different bead. There is some variability among beads and preparation, but on average beads move at micron/sub-micron per min speeds (0.2-1 &#181;m/min) under the standard conditions described here. (B) Representative graph showing evaluation of comet length (whole population of beads) over time. The slope of the linear correlation corresponds to the average displacement speed, in this case 0.24 &#181;m/min. (D) In the absence of capping activity (no capping protein or gelsolin), actin clouds form around beads, but comets do not form. All images are of 4.5 &#181;m diameter beads, epifluorescence imaging of fluorescent actin, scale bars = 5 &#181;m. <a href="https://www.jove.com/files/ftp_upload/64261/64261fig03large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>

Watch this Scientific Journal Video about Reconstitution of Actin-Based Motility with Commercially Available Proteins at JoVE.com

Reconstitution of Actin-Based Motility with Commercially Available Proteins

This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Many cell movements and shape changes and certain types of intracellular bacterial and organelle motility are driven by the biopolymer actin that forms a ...

reconstitution-actin-based-motility-with-commercially-available

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1.6K Views.  Sorbonne Université, Université Paris Cité. This protocol describes how to produce actin comets on the surfaces of beads using commercially available protein ingredients. Such systems mimic the protrusive structures found in cells, and can be used to examine physiological mechanisms of force production in a simplified way.

Video: Reconstitution of Actin-Based Motility with Commercially Available Proteins

Fabrication of Electrochemical-DNA Biosensors for the Reagentless Detection of Nucleic Acids, Proteins and Small Molecules

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. Many patients would be better served by rapid, bedside tests. To this end our laboratory and others have developed a versatile, reagentless 
biosensor platform that supports the quantitative, reagentless, electrochemical detection of nucleic acids (DNA, RNA), proteins (including antibodies) and small 
molecules analytes directly in unprocessed clinical and environmental samples. In this video, we demonstrate the preparation and use of several biosensors in this 
"E-DNA" class. In particular, we fabricate and demonstrate sensors for the detection of a target DNA sequence in a polymerase chain reaction mixture, an HIV-specific antibody and the drug cocaine. The preparation procedure requires only three hours of hands-on effort followed by an overnight incubation, and their use requires only minutes.

"E-DNA" sensors, reagentless, electrochemical biosensors that perform well even when challenged directly in blood and other complex matrices, have been adapted to the detection of a wide range of nucleic acid, protein and small molecule analytes. Here we present a general procedure for the fabrication and use of such sensors.

As medicine is currently practiced, doctors send specimens to a central laboratory for testing and thus must wait hours or days to 
receive the results. ...

Live-cell Imaging of Migrating Cells Expressing Fluorescently-tagged Proteins in a Three-dimensional Matrix

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound healing, tissue regeneration, and cancer metastasis, cells must navigate through complex, three-dimensional extracellular tissue. To better understand the mechanisms behind these biological processes, it is important to examine the roles of the proteins responsible for driving cell migration. Here, we outline a protocol to study the mechanisms of cell migration using the epithelial cell line (MDCK), and a three-dimensional, fibrous, self-polymerizing matrix as a model system. This optically clear extracellular matrix is easily amenable to live-cell imaging studies and better mimics the physiological, soft tissue environment. This report demonstrates a technique for directly visualizing protein localization and dynamics, and deformation of the surrounding three-dimensional matrix. 
Examination of protein localization and dynamics during cellular processes provides key insight into protein functions. Genetically encoded fluorescent tags provide a unique method for observing protein localization and dynamics. Using this technique, we can analyze the subcellular accumulation of key, force-generating cytoskeletal components in real-time as the cell maneuvers through the matrix. In addition, using multiple fluorescent tags with different wavelengths, we can examine the localization of multiple proteins simultaneously, thus allowing us to test, for example, whether different proteins have similar or divergent roles. Furthermore, the dynamics of fluorescently tagged proteins can be quantified using Fluorescent Recovery After Photobleaching (FRAP) analysis. This measurement assays the protein mobility and how stably bound the proteins are to the cytoskeletal network.
By combining live-cell imaging with the treatment of protein function inhibitors, we can examine in real-time the changes in the distribution of proteins and morphology of migrating cells. Furthermore, we also combine live-cell imaging with the use of fluorescent tracer particles embedded within the matrix to visualize the matrix deformation during cell migration. Thus, we can visualize how a migrating cell distributes force-generating proteins, and where the traction forces are exerted to the surrounding matrix. Through these techniques, we can gain valuable insight into the roles of specific proteins and their contributions to the mechanisms of cell migration.

Cellular processes such as cell migration have traditionally been studied on two-dimensional, stiff plastic surfaces. This report describes a technique for directly visualizing protein localization and analyzing protein dynamics in cells migrating in a more physiologically relevant, three-dimensional matrix.

Traditionally, cell migration has been studied on two-dimensional, stiff plastic surfaces. However, during important biological processes such as wound ...

Air Filter Devices Including Nonwoven Meshes of Electrospun Recombinant Spider Silk Proteins

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly repetitive protein has a molecular weight of 48kDa and is soluble in different solvents (hexafluoroisopropanol (HFIP), formic acid and aqueous buffers). eADF4(C16) provides a high potential for various technical applications when processed into morphologies such as films, capsules, particles, hydrogels, coatings, fibers and nonwoven meshes. Due to their chemical stability and controlled morphology, the latter can be used to improve filter materials. In this protocol, we present a procedure to enhance the efficiency of different air filter devices, by deposition of nonwoven meshes of electrospun recombinant spider silk proteins. Electrospinning of eADF4(C16) dissolved in HFIP results in smooth fibers. Variation of the protein concentration (5-25% w/v) results in different fiber diameters (80-1,100 nm) and thus pore sizes of the nonwoven mesh.
Post-treatment of eADF4(C16) electrospun from HFIP is necessary since the protein displays a predominantly &alpha;-helical secondary structure in freshly spun fibers, and therefore the fibers are water soluble. Subsequent treatment with ethanol vapor induces formation of water resistant, stable &beta;-sheet structures, preserving the morphology of the silk fibers and meshes. Secondary structure analysis was performed using Fourier transform infrared spectroscopy (FTIR) and subsequent Fourier self-deconvolution (FSD).
The primary goal was to improve the filter efficiency of existing filter substrates by adding silk nonwoven layers on top. To evaluate the influence of electrospinning duration and thus nonwoven layer thickness on the filter efficiency, we performed air permeability tests in combination with particle deposition measurements. The experiments were carried out according to standard protocols.

Spider silk fibers display extraordinary mechanical properties. Engineered Araneus diadematus Fibroin 4 (eADF4) can be processed into nonwoven meshes using electrospinning. Here, the eADF4 nonwoven meshes are used to improve the performance of air filtering devices.

Based on the natural sequence of Araneus diadematus Fibroin 4 (ADF4), the recombinant spider silk protein eADF4(C16) has been engineered. This highly ...

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Electric Cell-substrate Impedance Sensing for the Quantification of Endothelial Proliferation, Barrier Function, and Motility

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. To this end, cells are grown in special culture chambers on top of opposing, circular gold electrodes. A constant small alternating current is applied between the electrodes and the potential across is measured. The insulating properties of the cell membrane create a resistance towards the electrical current flow resulting in an increased electrical potential between the electrodes. Measuring cellular impedance in this manner allows the automated study of cell attachment, growth, morphology, function, and motility. Although the ECIS measurement itself is straightforward and easy to learn, the underlying theory is complex and selection of the right settings and correct analysis and interpretation of the data is not self-evident. Yet, a clear protocol describing the individual steps from the experimental design to preparation, realization, and analysis of the experiment is not available. In this article the basic measurement principle as well as possible applications, experimental considerations, advantages and limitations of the ECIS system are discussed. A guide is provided for the study of cell attachment, spreading and proliferation; quantification of cell behavior in a confluent layer, with regard to barrier function, cell motility, quality of cell-cell and cell-substrate adhesions; and quantification of wound healing and cellular responses to vasoactive stimuli. Representative results are discussed based on human microvascular (MVEC) and human umbilical vein endothelial cells (HUVEC), but are applicable to all adherent growing cells.

This protocol reviews Electric Cell-substrate Impedance Sensing, a method to record and analyze the impedance spectrum of adherent cells for the quantification of cell attachment, proliferation, motility, and cellular responses to pharmacological and toxic stimuli. Detection of endothelial permeability and assessment of cell-cell and cell-substrate contacts are emphasized.

Electric Cell-substrate Impedance Sensing (ECIS) is an in vitro impedance measuring system to quantify the behavior of cells within adherent cell layers. ...

Hydrogel Nanoparticle Harvesting of Plasma or Urine for Detecting Low Abundance Proteins

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers that exist in biological fluids cannot be easily detected with mass spectrometry or immunoassays because they are present in very low concentration, are labile, and are often masked by high-abundance proteins such as albumin or immunoglobulin. Bait containing poly(N-isopropylacrylamide) (NIPAm) based nanoparticles are able to overcome these physiological barriers. In one step they are able to capture, concentrate and preserve biomarkers from body fluids. Low-molecular weight analytes enter the core of the nanoparticle and are captured by different organic chemical dyes, which act as high affinity protein baits. The nanoparticles are able to concentrate the proteins of interest by several orders of magnitude. This concentration factor is sufficient to increase the protein level such that the proteins are within the detection limit of current mass spectrometers, western blotting, and immunoassays. Nanoparticles can be incubated with a plethora of biological fluids and they are able to greatly enrich the concentration of low-molecular weight proteins and peptides while excluding albumin and other high-molecular weight proteins. Our data show that a 10,000 fold amplification in the concentration of a particular analyte can be achieved, enabling mass spectrometry and immunoassays to detect previously undetectable biomarkers.

Several pathological biomarkers cannot be easily detected by current techniques because of their low concentration in biological fluids, the presence of degrading enzymes, and large amounts of high molecular weight proteins. Chemically functionalized hydrogel nanoparticles can harvest, preserve and concentrate low abundance proteins enabling the detection of previously undetectable biomarkers.

Novel biomarker discovery plays a crucial role in providing more sensitive and specific disease detection. Unfortunately many low-abundance biomarkers ...

Delivery of Proteins, Peptides or Cell-impermeable Small Molecules into Live Cells by Incubation with the Endosomolytic Reagent dfTAT

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the efficiency with which macromolecules penetrate the cytosolic space of cells. Recently, we have circumvented this problem by identifying the reagent dfTAT, a disulfide bond dimer of the peptide TAT labeled with the fluorophore tetramethylrhodamine. We have generated a fluorescently labeled dimer of the prototypical cell-penetrating peptide (CPP) TAT, dfTAT, which penetrates live cells and reaches the cytosolic space of cells with a particularly high efficiency. Cytosolic delivery of dfTAT is achieved in multiple cell lines, including primary cells. Moreover, delivery does not noticeably impact cell viability, proliferation or gene expression. dfTAT can deliver small molecules, peptides, antibodies, biologically active enzymes and a transcription factor. In this report, we describe the protocols involved in dfTAT synthesis and cellular delivery. The manuscript describes how to control the amount of protein delivered to the cytosolic space of cells by varying the amount of protein administered extracellularly. Finally, the current limitations of this new technology and steps involved in validating delivery are discussed. The described protocols should be extremely useful for cell-based assays as well as for the ex vivo manipulation and reprogramming of cells.

We describe how to deliver proteins and cell-impermeable small molecules into cultured mammalian cells by a simple co-incubation protocol with a reagent that causes endocytic organelles to become leaky.

Macromolecular delivery strategies typically utilize the endocytic pathway as a route of cellular entry. However, endosomal entrapment severely limits the ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

The Organoid Reconstitution Assay (ORA) for the Functional Analysis of Intestinal Stem and Niche Cells

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. Adult intestinal stem cells reside at the bottom of the crypts of Lieberk&#252;hn, are earmarked by expression of the Lgr5 gene, and preserve homeostasis through their characteristic high proliferative rate1. Throughout the small intestine, Lgr5+ stem cells are intermingled with specialized secretory cells called Paneth cells. Paneth cells secrete antibacterial compounds (i.e., lysozyme and cryptdins/defensins) and exert a controlling role on the intestinal flora. More recently, a novel function has been discovered for Paneth cells, namely their capacity to provide niche support to Lgr5+ stem cells through several key ligands as Wnt3, EGF, and Dll12.
When isolated ex vivo and cultured in the presence of specific growth factors and extracellular matrix components, whole intestinal crypts give rise to long-lived and self-renewing 3D structures called organoids that highly resemble the crypt-villus epithelial architecture of the adult small intestine3. Organoid cultures, when established from whole crypts, allow the study of self-renewal and differentiation of the intestinal stem cell niche, though without addressing the contribution of its individual components, namely the Lgr5+ and Paneth cells.
Here, we describe a novel approach to the organoid assay that takes advantage of the ability of Paneth and Lgr5+ cells to associate and form organoids when co-cultured. This approach, here referred to as &#34;organoid reconstitution assay&#34; (ORA), allows the genetic and biochemical modification of Paneth or Lgr5+ stem cells, followed by reconstitution into organoids. As such, it allows the functional analysis of the two main components of the intestinal stem cell niche.

The Organoid Reconstitution Assay ORA for the Functional Analysis of Intestinal Stem and Niche Cells

Intestinal organoid cultures are established from whole crypts and do not allow the analysis of self-renewal and differentiation in a cell-specific fashion. This protocol describes reconstitution of sorted stem (Lgr5+) and niche (Paneth) cells, which give rise to organoids while enabling their prior biochemical and genetic modification and functional analysis.

The intestinal epithelium is characterized by an extremely rapid turnover rate. In mammals, the entire epithelial lining is renewed within 4 - 5 days. ...