Name: preparation of keratin hydrolysate from chicken feathers and its application in cosmetics
Uploaded: 2017-11-27T14:00:00
Description: Watch this Scientific Journal Video about Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics at JoVE.com

This article was written with support of the project IGA/FT/2017/007 of Tomas Bata University in Zlin.

Volunteers were recruited among employees and students of our university. The method of selecting was conducted according to &#34;International Ethical Guidelines for Biomedical Research Involving Human Subjects. Council for International Organizations of Medical Sciences, Geneva (2002).&#34;&#160;KH is a common cosmetic ingredient used in hair-care products (shampoos, conditioners, etc.) and hence approval from the institutional review board is not required.
1. Process Chicken Feathers...

<ol>
	<li>United States Department of Agriculture - National Agricultural Statistics Services. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Poultry Slaughter, 2016 Summary. , (2016).</li><li>McGovern, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recycling+poultry+feathers:+more+bang+for+the+cluck.">Recycling poultry feathers: more bang for the cluck.</a> Environ.Health Perspect. 108 (8), A336-A339 (2000).</li><li>Gousterova, A., 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=Degradation+of+keratin+and+collagen+containing+wastes+by+newly+isolated+thermoactinomycetes+or+by+alkaline+hydrolysis.">Degradation of keratin and collagen containing wastes by newly isolated thermoactinomycetes or by alkaline hydrolysis.</a> Lett. Appl. Microbiol. 40 (5), 335-340 (2005).</li><li>Yamauchi, K., Yamauchi, A., Kusunoki, T., Khoda, A., Konishi, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Preparation+of+stable+aqueous+solution+of+keratins,+and+physiochemical+and+biodegradational+properties+of+films.">Preparation of stable aqueous solution of keratins, and physiochemical and biodegradational properties of films.</a> Biomed. Mater. Res. 31 (4), 439-444 (1996).</li><li>Schrooyen, P. M. M., Dijkstra, P. J., Oberthur, R. C., Bantjes, A., Feijen, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Partially+carboxymethylated+feather+keratins.+2.+Thermal+and+mechanical+properties+of+films.">Partially carboxymethylated feather keratins. 2. Thermal and mechanical properties of films.</a> J. Agric. Food Chem. 49 (1), 221-230 (2001).</li><li>Mark, H. F., Gaylord, N. G., Bikales, N. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Encyclopedia of Polymer Science Technology: vol. 8: Keratin to Modacrylic Fibers. , (1968).</li><li>Bertsch, A., Cello, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+biotechnological+process+for+treatment+and+recycling+poultry+feathers+as+a+feed+ingredient.">A biotechnological process for treatment and recycling poultry feathers as a feed ingredient.</a> Bioresour. Technol. 96 (15), 1703-1708 (2005).</li><li>Grazziotin, A., Pimentel, F. A., de Jong, E. V., Brandelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nutritional+improvement+of+feather+protein+by+treatment+with+microbial+keratinase.">Nutritional improvement of feather protein by treatment with microbial keratinase.</a> Animal Feed Sci. Technol. 126 (1-2), 135-144 (2006).</li><li>Brandelli, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Bacterial+keratinases:+useful+enzymes+for+bioprocessing+agroindustrial+wastes+and+beyond.">Bacterial keratinases: useful enzymes for bioprocessing agroindustrial wastes and beyond.</a> Food Bioprocess Technol. 1 (2), 105-116 (2008).</li><li>Gusta, R., Ramnani, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microbial+keratinases+and+their+prospective+applications:+an+overview.">Microbial keratinases and their prospective applications: an overview.</a> Appl.Microbiol. Biotechnol. 70 (1), 21-33 (2006).</li><li>Vasileva-Tonkova, E., Gousterova, A., Neshev, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ecologically+safe+method+for+improved+feather+wastes+biodegradation.">Ecologically safe method for improved feather wastes biodegradation.</a> International Biodeterior &amp; Biodegradation. 63 (8), 1008-1012 (2009).</li><li>Lod&#233;n, M., Barel, A. O., Paye, M., Maibach, H. I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrating+Substance.">Hydrating Substance.</a> Handbook of Cosmetic Science and Technology. , 107-119 (2009).</li><li>Teglia, A., Secchi, G., Goddard, E. D., Gruber, J. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chapter+9:+Proteins+in+Cosmetics.">Chapter 9: Proteins in Cosmetics.</a> Principles of Polymer Science and Technology in Cosmetics and Personal Care. , (1999).</li><li>Magdassi, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Delivery+systems+in+cosmetics.">Delivery systems in cosmetics.</a> Colloids and Surfaces A: Physicochem. Engin. Aspects. 123-124, 671-679 (1997).</li><li>Dahms, G., Jung, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Method+for+producing+a+protein+hydrolysate.">Method for producing a protein hydrolysate.</a> U.S. Patent. , (2014).</li><li>Pons, R., Carrera, I., Erra, P., Kunieda, G., Solans, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+preparation+methods+for+highly+concentrated+water-in-oil+emulsions.">Novel preparation methods for highly concentrated water-in-oil emulsions.</a> Colloids and Surfaces A: Physicochem. Engin. Aspects. 91 (3), 259-266 (1994).</li><li>Mokrejs, P., Hrncirik, J., Janacova, D., Svoboda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+of+keratin+waste+of+meat+industry.">Processing of keratin waste of meat industry.</a> Asian J. Chem. 24 (4), 1489-1494 (2012).</li><li>Mokrejs, P., Svoboda, P., Hrncirik, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Processing+poultry+feathers+into+keratin+hydrolysate+through+alkaline-enzymatic+hydrolysis.">Processing poultry feathers into keratin hydrolysate through alkaline-enzymatic hydrolysis.</a> Waste Manage. Res. 29 (3), 260-267 (2011).</li><li>Mokrejs, P., Krejci, O., Svoboda, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Producing+keratin+hydrolysates+from+sheep+wool.">Producing keratin hydrolysates from sheep wool.</a> Orient. J. Chem. 27 (4), 1303-1309 (2011).</li><li>Mokrejs, P., Krejci, O., Svoboda, P., Vasek, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Modeling+technological+conditions+for+breakdown+of+waste+sheep+wool.">Modeling technological conditions for breakdown of waste sheep wool.</a> Rasayan J. Chem. 4 (4), 728-735 (2011).</li><li>Polaskova, J., Pavlackova, J., Vltavska, P., Mokrejs, P., Janis, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Moisturizing+effect+of+topical+cosmetic+products+applied+to+dry+skin.">Moisturizing effect of topical cosmetic products applied to dry skin.</a> J. Cosmet. Sci. 64 (5), 329-340 (2013).</li><li>Polaskova, J., Pavlackova, J., Egner, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effect+of+vehicle+on+the+performance+of+active+moisturizing+substances.">Effect of vehicle on the performance of active moisturizing substances.</a> Skin Res. Technol. 21 (4), 403-412 (2015).</li><li>Verdier-S&#233;vrain, S., Bont&#233;, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+hydration:+a+review+on+its+molecular+mechanisms.">Skin hydration: a review on its molecular mechanisms.</a> J. Cosmet. Dermatol. 6 (2), 75-82 (2007).</li><li>Darlenski, R., Sassning, S., Tsankov, N., Fluhr, J. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Non-invasive+in+vivo+methods+for+investigation+of+the+skin+barrier+physical+properties.">Non-invasive in vivo methods for investigation of the skin barrier physical properties.</a> Eur. J. Pharm. Biopharm. 72 (2), 295-303 (2009).</li><li>Berardesca, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEMCO+guidance+for+assessment+of+stratum+corneum+hydration:+electrical+methods.">EEMCO guidance for assessment of stratum corneum hydration: electrical methods.</a> Skin Res. Technol. 3 (2), 126-132 (1997).</li><li>Rogiers, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=EEMCO+guidance+for+the+assessment+of+transepidermal+water+loss+in+cosmetic+sciences.">EEMCO guidance for the assessment of transepidermal water loss in cosmetic sciences.</a> Skin Pharmacol. Appl. Skin Physiol. 14 (2), 117-128 (2001).</li><li>Ali, S. M., Yosipovitch, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Skin+pH:+from+basic+science+to+basic+skin+care.">Skin pH: from basic science to basic skin care.</a> Acta Derm. Venereol. 93 (3), 261-267 (2013).</li><li>Agache, P., Humbert, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Measuring the Skin. , (2004).</li><li>Council for International Organizations of Medical Sciences. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> International Ethical Guidelines for Biomedical Research Involving Human Subjects. , (2002).</li><li>Ruland, 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=Transdermal+permeability+and+skin+accumulation+of+amino+acids.">Transdermal permeability and skin accumulation of amino acids.</a> Int. J. Pharm. 72 (2), 149-155 (1991).</li><li>Draelos, Z. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Therapeutic+moisturizers.">Therapeutic moisturizers.</a> Dermatol. Clin. 18 (4), 597-607 (2000).</li><li>Courage and Khazaka Electronic GmbH, Technical Charges. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Information and Operating Instructions for the Multi probe Adapter MPA and its Probe. , (2013).</li></ol>

The advantage of alkaline-enzymatic hydrolysis is that it can be modified according to future applications of KH. For example, in hair-care cosmetics applications where a lightly brownish color of a product is not an obstacle, a higher temperature in the hydrolysis can be applied leading to a higher yield of KH. In addition, the longer processing time during both stages of the technological procedure significantly affects the overall process efficiency &#8211; yield of KH rises to 85%.
The fin...

Slaughterhouses, the food industry, and the tanning industry annually produce immense amounts of solid keratin by-products &#8211; wool, feathers, bristles, hooves, claws, horns, and the like. According to latest statistical data, the total live weight of chickens, turkeys, ducks, and other slaughtered poultry in the USA is 62.5 billion pounds per year1; in the EU it is approximately 28.7 billion pounds per year. Considering that feathers make up to 8.5% of the total poultry weight, the USA alone annually produces approx. 5.3 billion pounds of waste feathers2.
Keratin is a protein exhibiting high chemical resistance because it is strongly cross-linked with disulfide bridges that render its processing difficult. Obtaining soluble products requires cleaving cross-links and possibly carrying out hydrolysis of the peptide bonds3. Cleavage of the disulfide bridges may proceed through a reaction of thiol anion according to the following pattern4,5:
Sa- + &#8211;SbSc&#8211; &#8596; &#8211;Sb- + &#8211;SaSc-
With a very high pH level, hydrolysis of the disulfide bridges also appears, in accordance with the pattern6
&#8211;SS- + OH- &#8594; &#8211;S- + &#8211;SOH
Under mild conditions (pH approx. 8), even sulfitolysis takes place according to the following pattern:
&#8211;SS&#8211; + HSO3- &#8594; &#8211;SH + &#8211;SSO3-
The most economical way of degrading keratin is microbial breakdown, which is characterized by mild reaction conditions during processing and high breakdown efficiency (approx. 90%)7,8. Keratinases are produced by some bacteria isolated from soil and keratin waste9. Microbial keratinases hydrolyze rigid and strongly cross-linked keratin structures10 and the resulting KH prepared is rich in soluble proteins, with no loss in essential amino acids detected in it11.
In order to incorporate a protein in cosmetic preparations (e.g., emulsions, lotions, and gels), the requirements ensure that such proteins are soluble in water, the given systems are transparent, and that re-aggregation of the peptides is avoided due to hydrophobic interactions. Therefore, a common practice is to apply hydrolysates of proteins, such as hydrolyzed collagen, elastin, and keratin. When adding hydrolysates into cosmetic emulsions, steps are taken to ensure that the hydrolysate is first dissolved in water. In some cases, it is desirable that the protein (or the hydrolysate) is soluble in alcohol or other organic solvents12.
KH is normally featured in shampoos, conditioners, lotions, and nutritive serums for hair, as well as mascaras, nail polish, and eye make-up agents. The KH effects declared usually include forming a protective film, smoothing the hair or nail structure, heightened plasticity and appearance of the treated formation, regulating the consistency of products, and encouraging the formation of foam13,14. It has also been shown that KH reduces surface tension, hence supplementation in cosmetics can facilitate reduction in the amount of emulsifier added to stabilize creams. KH limit the effects of irritation triggered by cleaning agents (surfactants) to the skin, eyes, and hair, thus reducing any potential side effects of cleaning agents on tissue (e.g., dehydration of the skin, hardness, and decreased barrier function of the skin). The high buffering capability of hydrolysates is also exploited to stabilize the pH of cosmetics; peptides of shorter length have a greater buffering effect15,16. Although KHs have become established as standard components in hair and nail cosmetics as well as being utilized in products for skin care, studies on the moisturizing effects of KH do not appear in contemporary literature.
Alkaline-enzymatic technology has been developed for processing keratin by-products into KH, and active testing is in process on the effects of a number of cosmetic additives17,18,19,20,21,22. The advantage of two-stage alkaline-enzymatic hydrolysis using microbial proteases for chicken feathers achieves high efficiency under mild reaction conditions and the quality of KH is very high in contrast to hydrolysis employed in strong acids or alkalis. In the first stage, feathers are incubated at a higher temperature in an alkaline environment, which partially disrupts the keratin structure and swells the feathers; after adjusting the pH, the feathers are hydrolyzed with a proteolytic enzyme under mild conditions in the second stage. The dialyzed KH possesses a high content of proteins.
The purposes of the method described here are processing poultry feathers into a KH through alkaline-enzymatic hydrolysis and testing the effect of moisturizing properties of KH applied to O/W cosmetic emulsion. The moisturizing properties are investigated by instrumental non-invasive methods in vivo. The most frequent methods for measuring skin hydration and barrier function of SC include measuring electrical properties of the skin (conductance or capacitance). Different methods for investigating SC hydration include near infrared multispectral imagining method (NIM), nuclear magnetic resonance spectroscopy, optical coherence tomography, or transient thermal transfer23. Barrier function of SC correlates to the TEWL of SC and it is measured by the ventilated chamber method, unventilated chamber method, and open chamber method24.
Properties of the model formulations are determined using the Multi Probe adapter MPA 5 with three types of probes. The first one, corneometer CM 825, measures skin hydration by assessing changes in the electrical capacity of the skin&#39;s surface; the measuring capacitor shows changes in capacitance of the skin surface in corneometric units. The corneometer gives only a relative assessment of skin hydration25. For TEWL, the second probe, tewameter TM 300, is used for measuring the density gradient of water evaporation (in an open chamber instrument based on Fick&#39;s diffusion law) from the skin indirectly by the two pairs of sensors (temperature and relative humidity) indicating the quantity of water being transported per a defined area and period of time (g/m2/h). This method can detect even the slightest disruption of skin barrier function26. Skin pH is one indicator of barrier and anti-microbial function of the SC27. The acidity of the skin mantle was measured by a (third) skin PH 905 probe connected to the MPA 5 station. This specially designed probe consists of a flat-topped glass electrode for full skin contact, connected to a voltmeter. The system measures potential changes due to the activity of hydrogen cations surrounding the very thin layer of semi-solid forms measured at the top of the probe. The changes in voltage are displayed as pH28.
We present experiments divided into three sections: (1) Preparation of KH from chicken feathers by two-stage alkaline-enzymatic hydrolysis and its purification by dialysis (removing salts and low-molecular fractions), (2) Preparation of cosmetic formulations containing 2, 4, and 6% KH, and (3) Testing the properties of KH by measuring skin hydration, TEWL, and skin pH. Testing was carried out on 10 women with the mean age of 27.2 years and on 10 men with the mean age of 26.2 years. The method of selecting the volunteers and the testing itself were conducted in accordance with international ethical principles of bio-medical research utilizing human subjects29; all persons gave their informed consent prior to inclusion in the study. Before testing commenced, the volunteers were asked to complete a questionnaire on their health status. The volunteers committed to avoid applying any cosmetic product to the test sites and surrounding regions during the 24 h prior to and during the test period; furthermore, they were only permitted brief evening washes with running water.

<table>
	<tbody>
		<tr>
			<td>Material or chemicals</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>LIPEX 100T</td>
			<td>Novozymes</td>
			<td>LJP30020</td>
			<td>Lipex - enzyme produced by submerged fermentation of a genetically-modified microorganism, activity 100 KLU/g</td>
		</tr>
		<tr>
			<td>Savinase Ultra 16L</td>
			<td>Novozymes</td>
			<td>PXN40001</td>
			<td>Savinase - enzyme produced by submerged fermentation of a genetically-modified microorganism, activity 16 KNPU-S/g</td>
		</tr>
		<tr>
			<td>Potassium hydroxide, KOH</td>
			<td>Sigma-Aldrich</td>
			<td>302510289</td>
			<td>Potassium hydroxide, KOH, 97,0 %, Mr 56,11</td>
		</tr>
		<tr>
			<td>Phosphoric acid solution, H3PO4</td>
			<td>Sigma-Aldrich</td>
			<td>W290017</td>
			<td>Phosphoric acid solution, H3PO4, 85 wt. % concentration in water, Mr 98,00</td>
		</tr>
		<tr>
			<td>Sodium chloride physiological solution</td>
			<td>Sigma-Aldrich</td>
			<td>52455</td>
			<td>Tablets of BioUltra NaCl physiological solution; 1 tablet in 1000 mL of water yields 0.9 % NaCl</td>
		</tr>
		<tr>
			<td>Sodium hydroxide, NaOH</td>
			<td>Penta s.r.o.</td>
			<td>40216</td>
			<td>Sodium hydroxide, NaOH, 97,0 %, Mr 40,00</td>
		</tr>
		<tr>
			<td>AmiFarm (Cremor base-A)</td>
			<td>Fagron</td>
			<td>608425</td>
			<td>Hydrophilic oil in water (O/W) cream base; the composition: aqua, paraffin, paraffin liquid, cetearyl alkohol, Laureth 4, sodium hydroxide, carbomer, methylparaben, propylparaben.</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>IKA EUROSTAR POWER control-visc stirrers</td>
			<td>IKA-labortechnik</td>
			<td>Z404020</td>
			<td>Digital laboratory stirrer, for tasks up to the high viscosity range, 230V, 1/cs</td>
		</tr>
		<tr>
			<td>IKA Propeller stirrer, 3-bladed</td>
			<td>IKA-labortechnik</td>
			<td>R 1381</td>
			<td>Propeller stirrer, 3-bladed, stirrer &#216;: 45 mm, shaft &#216;: 8 mm, shaft length: 350 mm</td>
		</tr>
		<tr>
			<td>Dialysis tubing closures</td>
			<td>Sigma-Aldrich</td>
			<td>Z371017-10EA</td>
			<td>Dialysis tubing closures, red, size 110 mm</td>
		</tr>
		<tr>
			<td>Dialysis tubing cellulose membrane</td>
			<td>Sigma-Aldrich</td>
			<td>D9402-100FT</td>
			<td>Dialysis tubing cellulose membrane, average flat width 76 mm (3.0 in.)</td>
		</tr>
		<tr>
			<td>DOMO Pot with stailess, LCD</td>
			<td>DOMO Elektronic</td>
			<td>DO42325PC</td>
			<td>Preserving boiler stainless steel, 2000 W, 27-L container (diameter 37 cm, height 30 cm), temperature control 30-100 &#176; C, operation LCD display</td>
		</tr>
		<tr>
			<td>Hettich zentrifugen Universal 32</td>
			<td>Gemini bv</td>
			<td>2770 GS1R</td>
			<td>Mid bench centrifuge, speed 18000 rpm</td>
		</tr>
		<tr>
			<td>LT 3 shaking device</td>
			<td>Fischer Scientific</td>
			<td>6470.0002</td>
			<td>Orbital shaking device</td>
		</tr>
		<tr>
			<td>KERN 440-47N</td>
			<td>Kern</td>
			<td>440-47N</td>
			<td>Laboratory balance</td>
		</tr>
		<tr>
			<td>KERN 770</td>
			<td>Kern</td>
			<td>770 -N</td>
			<td>Laboratory analytical balance</td>
		</tr>
		<tr>
			<td>VENTICELL 222 - Komfort</td>
			<td>BMT, MMM Group</td>
			<td>C 131749</td>
			<td>Drying oven, temperature control 30-100 &#176; C, air circulation control</td>
		</tr>
		<tr>
			<td>Vacucell 55 - EVO</td>
			<td>BMT, MMM Group</td>
			<td>B 050328</td>
			<td>Vacuum drying oven, temperature control 30-100 &#176; C</td>
		</tr>
		<tr>
			<td>PULVERISETTE 19</td>
			<td>Fritsch</td>
			<td>19.1030.00</td>
			<td>Universal cutting mill, rotor with V-cutting edges and fixed knives</td>
		</tr>
		<tr>
			<td>Multi Probe Adapter System MPA 5</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td>10225237</td>
			<td>MPA 5 Station - equipment for measurement hydratation, TEWL and pH</td>
		</tr>
		<tr>
			<td>Skin pH-meter PH 905 probe</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe to specifically measure the pH on the skin surface or the scalp</td>
		</tr>
		<tr>
			<td>Corneometer CM 825 probe</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe to determine the hydration level of the skin surface (Stratum corneum).</td>
		</tr>
		<tr>
			<td>Tewameter TM 300</td>
			<td>Courage &#38; Kazaka Electronic</td>
			<td></td>
			<td>Probe for the assessment of the transepidermal water loss (TEWL)</td>
		</tr>
		<tr>
			<td>Heidolph RZR 2020</td>
			<td>Heidolph</td>
			<td>13-225-007-03-1</td>
			<td>Overhead stirrer, mechanical speed setting and stepless transmission; speed range 40-2000 rpm</td>
		</tr>
		<tr>
			<td>Heidolph mechanical stirrer BR 10</td>
			<td>Heidolph</td>
			<td>Z336688-1EA</td>
			<td>Blade impeller crossed stirrer</td>
		</tr>
		<tr>
			<td>Fagor FS 12</td>
			<td>Fagor</td>
			<td>BTT-138</td>
			<td>Laboratory refrigerator with freezer space</td>
		</tr>
		<tr>
			<td>WTW bench pH/mV meter</td>
			<td>WTW</td>
			<td>Z313165</td>
			<td>High-performance bench pH and pH/conductivity meters for routine and high precision laboratory measurements in research or quality control laboratories</td>
		</tr>
		<tr>
			<td>Container</td>
			<td>RPC Superfos</td>
			<td></td>
			<td>13-L plastic bucket, diameter 26 cm, height 26 cm</td>
		</tr>
		<tr>
			<td>Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr>
			<td>Software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Microsoft Office 2010</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>C+K software</td>
			<td colspan="2">Courage and Khazaka Electronic GmbH</td>
			<td>MPA 5 station operating software</td>
		</tr>
	</tbody>
</table>

preparation of keratin hydrolysate from chicken feathers and its application in cosmetics

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for skin-care cosmetics. The goals of the protocol are: (1) to process chicken feathers into KH by alkaline-enzymatic hydrolysis and purify it by dialysis, and (2) to test if adding KH into an ointment base (OB) increases hydration of the skin and improves skin barrier function by diminishing transepidermal water loss (TEWL). During alkaline-enzymatic hydrolysis feathers are first incubated at a higher temperature in an alkaline environment and then, under mild conditions, hydrolyzed with proteolytic enzyme. The solution of KH is dialyzed, vacuum dried, and milled to a fine powder. Cosmetic formulations comprising from oil in water emulsion (O/W) containing 2, 4, and 6 weight% of KH (based on the weight of the OB) are prepared. Testing the moisturizing properties of KH is carried out on 10 men and 10 women at time intervals of 1, 2, 3, 4, 24, and 48 h. Tested formulations are spread at degreased volar forearm sites. The skin hydration of stratum corneum (SC) is assessed by measuring capacitance of the skin, which is one of the most world-wide used and simple methods. TEWL is based on measuring the quantity of water transported per a defined area and period of time from the skin. Both methods are fully non-invasive. KH makes for an excellent occlusive; depending on the addition of KH into OB, it brings about a 30% reduction in TEWL after application. KH also functions as a humectant, as it binds water from the lower layers of the epidermis to the SC; at the optimum KH addition in the OB, up to 19% rise in hydration in men and 22% rise in women occurs.

The KH prepared according to procedure presented here (see Figure 2) is yellow in color, easily soluble in water with high protein content (inorganic solids represent &#60;2.0%); the pH of the 1.0% solution of KH is 5.3, and fulfils the requirements for cosmetic-grade hydrolysates. The yield of KH from 50 g raw material is approx. 30%. The molecular weight distribution of KH was determined by SDS-PAGE and is shown in Figure 3.</p...

Watch this Scientific Journal Video about Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics at JoVE.com

Preparation of Keratin Hydrolysate from Chicken Feathers and Its Application in Cosmetics

The goal of the protocol is to prepare keratin hydrolysate from chicken feathers by alkaline-enzymatic hydrolysis and to test whether adding keratin hydrolysate into a cosmetics ointment base improves skin barrier function (heightening hydration and diminishing transepidermal water loss). Tests are conducted on men and woman volunteers.

Keratin hydrolysates (KHs) are established standard components in hair cosmetics. Understanding the moisturizing effects of KH is advantageous for ...

The overall goal of this procedure is to prepare keratin hydrolysate from chicken feathers by alkaline enzymatic hydrolysis, and to test whether adding of keratin hydrolysate into a cosmetics ointment base improves skin barrier function. This method can help answer questions in the slaughterhouse keratin byproducts field such as utilization of chicken poultry feathers for preparation of keratin hydrolysate, which can be used in cosmetic formulations. The main advantage of this technique is that keratin hydrolysate is a high-quality and acts as a very good occlusive and humectant in cosmetic formulations.Demonstrating the procedure of applying tested formulations at each spot of the four arm sides will be Jana Pavlackova, a co-author of this publication. To begin, grind 50 grams of dried feathers in a cutting mill into a final fineness of 1.0 millimeters. Next, degrease the feathers in a stainless steel 27-liter boiler container with temperature control.Mix the feathers with water preheated up to 40 plus or minus two degrees celsius in a weight ratio of one to 75. Add a lipolytic enzyme in a dose of 1.5 to 2.0%and gently stir the contents with an overhead stirrer for five minutes. Adjust the mixture pH to 9.0 plus or minus 0.2 by adding 1%sodium hydroxide or 1%phosphoric acid solution.This is the pH value corresponding to the maximum activity of the lipolytic enzyme. After stirring the mixture for five minutes with an overhead stirrer, check the pH level using a laboratory bench pH/mV meter and readjust the pH level. Then, gently stir the mixture with an overhead stirrer for 24 hours at 40 plus or minus 0.5 degrees celsius.Filter the mixture through a fine sieve and wash the degreased feathers with a stream of fresh running cold water. Then, dry the feathers on a flat plate at 50 degrees celsius in a drying chamber overnight. The next day, perform the first stage of the chicken feather hydrolysis.Mix the feathers with 0.3%potassium hydroxide water solution in a weight ratio of one to 50, and gently stir with an overhead stirrer at 60 plus or minus 0.5 degrees celsius for 24 hours. After finishing the first stage of hydrolysis, adjust the pH of the mixture to the level corresponding to the maximum activity of the proteolytic enzyme with 10%phosphoric acid, which in this case, is a level of 9.0 plus or minus 0.2. Perform the second stage of chicken feather hydrolysis.Add the proteolytic enzyme to the mixture in a dose of 5.0%Gently stir the mixture with an overhead stirrer at 60 plus or minus 0.5 degrees celsius for eight hours. Then, heat the mixture in the same stainless steel 27-liter boiler container to the boiling point and boil for 10 minutes to inactivate the enzyme. Separate the solution of KH from the undissolved remnant by filtering it through low-density filter paper on a Buchner funnel with slight vacuum pressure.Pour 400 milliliters of the KH solution into 12K molecular weight cutoff dialysis tubing in order to remove small peptides and salts. Dialyze the solutions against four liters of distilled water in a plastic bucket for 80 hours at room temperature. Change the distilled water after 18, 36, and 60 hours.Cast the dialyzed solution of KH on an antiadhesive plate at a ratio of 500 milliliters to a 1, 000-square-centimeter plate area. Vacuum dry the solution to a thin film at 40 plus or minus 0.5 degrees celsius overnight. After scraping the dried film, grind it to form a fine powder and keep it in a closed vessel in a desiccator.Next, prepare cosmetic formulations containing two, four, and 6%KH in accordance with the base weight of the ointment. To do so, weigh the amount of KH powder into a polyethylene vessel. Then, add the ointment base at an amount that ensures the total weight of the formulation equals 50 grams.Homogenize the mixture with a three-bladed laboratory blender for 10 minutes at 134.16 g before mixing using a mechanical overhead stirrer. Maintain the prepared formulations at five plus or minus one degree celsius, and warm them at room temperature for two hours prior to use. Place five strips of filter paper into a physiological solution of 0.90%sodium chloride and leave them for approximately one minute in the solution.Apply two strips to the inner side of the right forearm, and three to the inner side of the left forearm, and fix them for four hours with adhesive plasters. After four hours, remove the strips and demark the areas with a permanent pen. Prepare to apply 0.1 milliliters of the tested formulations at each spot of the degreased forearm sites.On the left forearm, do not add anything to the first site as it is the control. Then, apply the ointment base only to the second site. Use syringes and spread it over the entire marked surface.Similarly, spread the ointment base plus 2%KH onto the third site. Finally, apply the ointment base plus 4%KH and ointment base plus 6%KH to the right arm. Measure each sample at each site and each interval.Take five readings with the skin hydration meter probe for skin hydration, 15 readings with the tool meter probe for skin transepidermal water loss, and one reading with the skin pH meter probe for skin pH. Process the resulting readings as described in the text protocol. Shown here is a representative photograph of keratin hydrolysate showing its yellow color.Keratin hydrolysate is easily soluble in water, has high protein content, and fulfills requirements for cosmetic-grade hydrolysates. Electrophoretic profiles of low molecular protein standard, high molecular protein standard, and keratin hydrolysate, show that keratin hydrolysate has wide molecular weight heterogeneity. Once mastered, this technique can be done in 150 hours if it is done properly.After watching this video you should have a good understanding of how to prepare keratin hydrolysate and how to apply it in cosmetic formulations. After its development, this technique paved the way for researchers in the field of protein byproduct treatment to explore utilization of chicken feathers in cosmetic industry.

preparation-keratin-hydrolysate-from-chicken-feathers-its-application

Research

JoVE Journal

Biochemistry

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from the oxidation of reduced compounds such as nicotinamide adenine dinucleotide (NADH) and flavin adenine dinucleotide (FADH2). The complete annotation of the Arabidopsis thaliana genome has established it as the most widely used plant model system, and thus the need to purify mitochondria from a variety of organs (leaf, root, or flower) is necessary to fully utilize the tools that are now available for Arabidopsis to study mitochondrial biology. Mitochondria are isolated by homogenization of the tissue using a variety of approaches, followed by a series of differential centrifugation steps producing a crude mitochondrial pellet that is further purified using continuous colloidal density gradient centrifugation. The colloidal density material is subsequently removed by multiple centrifugation steps. Starting from 100 g of fresh leaf tissue, 2 - 3 mg of mitochondria can be routinely obtained. Respiratory experiments on these mitochondria display typical rates of 100 - 250 nmol O2 min-1 mg total mitochondrial protein-1 (NADH-dependent rate) with the ability to use various substrates and inhibitors to determine which substrates are being oxidized and the capacity of the alternative and cytochrome terminal oxidases. This protocol describes an isolation method of mitochondria from Arabidopsis thaliana leaves using continuous colloidal density gradients and an efficient respiratory measurements of purified plant mitochondria.

Isolation and Respiratory Measurements of Mitochondria from Arabidopsis thaliana

As mitochondria are only a small percentage of the plant cell, they need to be purified for a range of studies. Mitochondria can be isolated from a variety of plant organs by homogenization, followed by differential and density gradient centrifugation to obtain a highly purified mitochondrial fraction.

Mitochondria are essential organelles involved in numerous metabolic pathways in plants, most notably the production of adenosine triphosphate (ATP) from ...

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine (DOPA) and Its Application to Protein Conjugation

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical properties, the amino acid has multiple uses in biochemical applications. This report describes a protocol for the genetic incorporation of biosynthesized DOPA and its application to protein conjugation. DOPA is biosynthesized by a tyrosine phenol-lyase (TPL) from catechol, pyruvate, and ammonia, and the amino acid is directly incorporated into proteins by the genetic incorporation method using an evolved aminoacyl-tRNA and aminoacyl-tRNA synthetase pair. This direct incorporation system efficiently incorporates DOPA with little incorporation of other natural amino acids and with better protein yield than the previous genetic incorporation system for DOPA. Protein conjugation with DOPA-containing proteins is efficient and site-specific and shows its usefulness for various applications. This protocol provides protein scientists with detailed procedures for the efficient biosynthesis of mutant proteins containing DOPA at desired sites and their conjugation for industrial and pharmaceutical applications.

Genetic Incorporation of Biosynthesized L-dihydroxyphenylalanine DOPA and Its Application to Protein Conjugation

Here, we present a protocol for the genetic incorporation of L-dihydroxyphenylalanine biosynthesized from simple starting materials and its application to protein conjugation.

L-dihydroxyphenylalanine (DOPA) is an amino acid found in the biosynthesis of catecholamines in animals and plants. Because of its particular biochemical ...

Isolation of Lipoprotein Particles from Chicken Egg Yolk for the Study of Bacterial Pathogen Fatty Acid Incorporation into Membrane Phospholipids

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the majority of exogenous fatty acids are present within host lipoprotein particles. Uncertainty remains as to the reservoirs of host fatty acids and the mechanisms by which bacteria extract fatty acids from the lipoprotein particles. In this work, we describe protocols for enrichment of low-density lipoprotein (LDL) particles from chicken egg yolk and determining whether LDLs serve as fatty acid reservoirs for S. aureus. This method exploits unbiased lipidomic analysis and chicken LDLs, an effective and economical model for the exploration of interactions between LDLs and bacteria. The analysis of S. aureus integration of exogenous fatty acids from LDLs is performed using high-resolution/accurate mass spectrometry and tandem mass spectrometry, enabling the characterization of the fatty acid composition of the bacterial membrane and unbiased identification of novel combinations of fatty acids that arise in bacterial membrane lipids upon exposure to LDLs. These advanced mass spectrometry techniques offer an unparalleled perspective of fatty acid incorporation by revealing the specific exogenous fatty acids incorporated into the phospholipids. The methods outlined here are adaptable to the study of other bacterial pathogens and alternative sources of complex fatty acids.

This method provides a framework for studying incorporation of exogenous fatty acids from complex host sources into bacterial membranes, particularly Staphylococcus aureus. To achieve this, protocols for the enrichment of lipoprotein particles from chicken egg yolk and subsequent fatty acid profiling of bacterial phospholipids utilizing mass spectrometry are described.

Staphylococcus aureus and other Gram-positive pathogens incorporate fatty acids from the environment into membrane phospholipids. During infection, the ...

Application of Biolayer Interferometry (BLI) for Studying Protein-Protein Interactions in Transcription

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA is a novel transcription activator found specifically in the obligate intracellular bacterial pathogen Chlamydia. Protein pulldown assays using affinity beads have revealed that GrgA binds two &#963; factors, namely &#963;66 and &#963;28, which recognize different sets of promoters for genes whose products are differentially required at developmental stages. We have used BLI to confirm and further characterize the interactions. BLI demonstrates several advantages over pulldown: 1) It reveals real-time association and dissociation between binding partners, 2) It generates quantitative kinetic parameters, and 3) It can detect bindings that pulldown assays often fail to detect. These characteristics have enabled us to deduce the physiological roles of GrgA in gene expression regulation in Chlamydia, and possible detailed interaction mechanisms. We envision that this relatively affordable technology can be extremely useful for studying transcription and other biological processes.

Application of Biolayer Interferometry BLI for Studying Protein-Protein Interactions in Transcription

Interactions of transcription factors (TFs) with the RNA polymerase are usually studied using pulldown assays. We apply a Biolayer Interferometry (BLI) technology to characterize the interaction of GrgA with the chlamydial RNA polymerase. Compared to pulldown assays, BLI detects real-time association and dissociation, offers higher sensitivity, and is highly quantitative.

A transcription factor&#160;(TF) is a protein that regulates gene expression by interacting with the RNA polymerase, another TF, and/or template DNA. GrgA ...

Preparation of High-Quality Fermented Fish Product

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures were: (1) pretreatment of farmed sturgeon including decapitation, evisceration, skinning-off, cleaning and cutting; (2) marinating fish cubes in 6-12% (w/v) salt solution (1:1, fish cube mass to solution volume); (3) drying fish cubes to a water content of 50-60% by hot air (40-60 &#176;C) or by vacuum; (4) fermentation involving inoculating fish cubes with 0.4-1.6% (w/w) S. cerevisiae in flavor solution to fish cubes and fermenting at 25-35 &#176;C for 6-10 h; (5) sealing fish cubes in vacuum packages with marinating and fermenting solutions; (6) sterilizing at 115-121 &#176;C for 10-20 min. The sturgeon meat product prepared by this method has delicious taste which is mellow and thick, has various types and large amounts of volatile flavor compounds such as alcohols and esters which could mask musty and unpleasant odor from fish, has moderate salt content but good texture properties such as high springiness, gumminess and chewiness, and has bright russet color and attractive appearance. This new technique could also be applied in the processing of other fish to provide convenient fish snack foods which could be stored at room temperature. It is appropriate for both marine and freshwater fish.

The goal of the protocol is to provide an industrialized fish fermentation technique based on inoculation of Saccharomyces cerevisiae.

This protocol provides a method for preparation of industrialized fermented fish product with sturgeon (Aquilaria sinensis) meat product. The procedures ...

JUMPn: A Streamlined Application for Protein Co-Expression Clustering and Network Analysis in Proteomics

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. However, deriving biological insights from such valuable datasets is challenging. Here we introduce a systems biology-based software JUMPn, and its associated protocol to organize the proteome into protein co-expression clusters across samples and protein-protein interaction (PPI) networks connected by modules (e.g., protein complexes). Using the R/Shiny platform, the JUMPn software streamlines the analysis of co-expression clustering, pathway enrichment, and PPI module detection, with integrated data visualization and a user-friendly interface. The main steps of the protocol include installation of the JUMPn software, the definition of differentially expressed proteins or the (dys)regulated proteome, determination of meaningful co-expression clusters and PPI modules, and result visualization. While the protocol is demonstrated using an isobaric labeling-based proteome profile, JUMPn is generally applicable to a wide range of quantitative datasets (e.g., label-free proteomics). The JUMPn software and protocol thus provide a powerful tool to facilitate biological interpretation in quantitative proteomics.

We present a systems biology tool JUMPn to perform and visualize network analysis for quantitative proteomics data, with a detailed protocol including data pre-processing, co-expression clustering, pathway enrichment, and protein-protein interaction network analysis.

With recent advances in mass spectrometry-based proteomics technologies, deep profiling of hundreds of proteomes has become increasingly feasible. ...

Exploring Biomolecular Interaction Between the Molecular Chaperone Hsp90 and Its Client Protein Kinase Cdc37 using Field-Effect Biosensing Technology

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. Biomolecules such as proteins, carbohydrates, vitamins, fatty acids, nucleic acids, and enzymes are fundamental building blocks of living beings; they assemble into complex networks in biosystems to synchronize a myriad of life events. Proteins typically utilize complex interactome networks to carry out their functions; hence it is mandatory to evaluate such interactions to unravel their importance in cells at both cellular and organism levels. Toward this goal, we introduce a rapidly emerging technology, field-effect biosensing (FEB), to determine specific biomolecular interactions. FEB is a benchtop, label-free, and reliable biomolecular detection technique to determine specific interactions and uses high-quality electronic-based biosensors. The FEB technology can monitor interactions in the nanomolar range due to the biocompatible nanomaterials used on its biosensor surface. As a proof of concept, the protein-protein interaction (PPI) between heat shock protein 90 (Hsp90) and cell division cycle 37 (Cdc37) was elucidated. Hsp90 is an ATP-dependent molecular chaperone that plays an essential role in the folding, stability, maturation, and quality control of many proteins, thereby regulating multiple vital cellular functions. Cdc37 is regarded as a protein kinase-specific molecular chaperone, as it specifically recognizes and recruits protein kinases to Hsp90 to regulate their downstream signal transduction pathways. As such, Cdc37 is considered a co-chaperone of Hsp90. The chaperone-kinase pathway (Hsp90/Cdc37 complex) is hyper-activated in multiple malignancies promoting cellular growth; therefore, it is a potential target for cancer therapy. The present study demonstrates the efficiency of FEB technology using the Hsp90/Cdc37 model system. FEB detected a strong PPI between the two proteins (KD values of 0.014 &#181;M, 0.053 &#181;M, and 0.072 &#181;M in three independent experiments). In summary, FEB is a label-free and cost-effective PPI detection platform, which offers fast and accurate measurements.

Field-effect biosensing (FEB) is a label-free technique for detecting biomolecular interactions. It measures the electric current through the graphene biosensor to which the binding targets are immobilized. The FEB technology was used to evaluate biomolecular interactions between Hsp90 and Cdc37 and a strong interaction between the two proteins was detected.

Biomolecular interactions play versatile roles in numerous cellular processes&#160;by regulating and coordinating functionally relevant biological events. ...

Advancing High-Resolution Imaging of Virus Assemblies in Liquid and Ice

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It is extremely desirable to pair high-resolution cryo-EM information with dynamic observations as many events occur at rapid timescales - in the millisecond range or faster. Improved knowledge of flexible structures can also assist in the design of novel reagents to combat emerging pathogens, such as SARS-CoV-2. More importantly, viewing biological materials in a fluid environment provides a unique glimpse of their performance in the human body. Presented here are newly developed methods to investigate the nanoscale properties of virus assemblies in liquid and vitreous ice. To accomplish this goal, well-defined samples were used as model systems. Side-by-side comparisons of sample preparation methods and representative structural information are presented. Sub-nanometer features are shown for structures resolved in the range of ~3.5-&#197;-10 &#197;. Other recent results that support this complementary framework include dynamic insights of vaccine candidates and antibody-based therapies imaged in liquid. Overall, these correlative applications advance our ability to visualize molecular dynamics, providing a unique context for their use in human health and disease.

Here protocols are described to prepare virus assemblies suitable for liquid-EM and cryo-EM analysis at the nanoscale using transmission electron microscopy.

Interest in liquid-electron microscopy (liquid-EM) has skyrocketed in recent years as scientists can now observe real-time processes at the nanoscale. It ...

Coating CryoEM Grids with Monolayer Graphene to Improve Cryo-Sample Preparation

Application of Monolayer Graphene to Cryo-Electron Microscopy Grids for High-resolution Structure Determination

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to remove excess liquid and rapidly frozen in a roughly 20-100 nm thick layer of vitreous ice, suspended across roughly 1 &#181;m wide foil holes. The resulting sample is imaged using cryogenic transmission electron microscopy, and after image processing using suitable software, near-atomic resolution structures can be determined. Despite cryoEM&#39;s widespread adoption, sample preparation remains a severe bottleneck in cryoEM workflows, with users often encountering challenges related to samples behaving poorly in the suspended vitreous ice. Recently, methods have been developed to modify cryoEM grids with a single continuous layer of graphene, which acts as a support surface that often increases particle density in the imaged area and can reduce interactions between particles and the air-water interface. Here, we provide detailed protocols for the application of graphene to cryoEM grids and for rapidly assessing the relative hydrophilicity of the resulting grids. Additionally, we describe an EM-based method to confirm the presence of graphene by visualizing its characteristic diffraction pattern. Finally, we demonstrate the utility of these graphene supports by rapidly reconstructing a 2.7 &#197; resolution density map of a Cas9 complex using a pure sample at a relatively low concentration.

Author Spotlight: Enhancing CryoEM Sample Preparation Using Graphene Monolayer on Microscopy Grids 

The application of support layers to cryogenic electron microscopy (cryoEM) grids can increase particle density, limit interactions with the air-water interface, reduce beam-induced motion, and improve the distribution of particle orientations. This paper describes a robust protocol for coating cryoEM grids with a monolayer of graphene for improved cryo-sample preparation.

In cryogenic electron microscopy (cryoEM), purified macromolecules are applied to a grid bearing a holey carbon foil; the molecules are then blotted to ...