המחברים מבקשים להודות ל: ד&quot;ר פרנק ג&#39;יאנג, ד&quot;ר דוד לין, ד&quot;ר ברנרד Yurke וד&quot;ר עודאי Chippada על תרומתם בפיתוח טכנולוגית ג&#39;ל DNA; ד&quot;ר Norell Hadzimichalis, סמיט השאה, קימברלי פיטרמן, רוברט Arter להערות והעריכות של כתב היד הזה שלהם; מקורות מימון כולל ועדת ניו ג&#39;רזי על חוט השדרה המחקר (גרנט # 07Â-019-SCR1, מעבדת המאיץ הלאומי) וניו ג&#39;רזי Neuroscience מכון (MLP); ומוציאים לאור של הנדסת רקמות, חלק א &#39;לאישור להדפיס מחדש איורים 2 ו 4 וBiomaterials רשות להדפיס מחדש את איור 3.

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יש לי המחברים אין לחשוף.

היכולת של ג&#39;לי DNA כדי לרכך או להקשיח לפני ואחרי הידבקות התא הופכת אותם למודל אידיאלי כדי ללמוד את התפקיד של קשיחות רקמה דינמית על תפקוד תא. כל שלושה העיצובים שנעשו שימוש במחקרים מכאניים וביולוגיים. עם זאת, יש את כל שלושת העיצובים גמישויות דומות בשיעורי crosslink שונים, ה?...

מצעים סטטי ודינמיים שתי קטגוריות של חומרים ביולוגיים שפותחו כדי לחקור את ההשפעות של גמישות רקמות או נוקשות בתפקוד תא. מצעי סטטי אינם מסוגלים לשנות את התכונות הפיזיות שלהם אחרי שהם מפוברקים ו / או פעם תאים מצופה. Polyacrylamide ג&#39;לים (הרשות הפלסטינית) היו מצעים הראשונים דו ממדים, סטטי שהיו מסונתזים לחקירות mechanobiology 5,17. ג&#39;לי הרשות הפלסטינית הם קל להכנה, זול, תכליתי, והוא יכול להיות מפוברק עם מגוון רחב של moduli אלסטי. למרות יתרונות הטכניים אלה הופכים את הרשות הפלסטינית ג&#39;לי מצע נפוץ מיושם, מצעי סטטי אינם מעידים על האופי הדינמי של מטריצת חוץ תאית (ECM) וסביבה סלולרית המקיפה in vivo. לדוגמא, ECM עובר שינויי נוקשות כתוצאה מפגיעה, פיתוח, או מחלה. מצעים דינמיים ולכן הם העדיפו כמודלים מצע מחקה רקמות במחקרי mechanobiology <sup&gt; 22,24,25. סינטטי רב, דו ממדי חומרים ביולוגיים טבעיים,, תלת ממדים, סטטי, ודינמיים פותחו כדי לחקות רקמות נוקשות 1,3,6,16,23,26. כמה מצעים דינמיים דורשים חום, UV, זרם חשמלי, יונים, ושינויי pH לשנות התכונות מכאניות שלהם 2,4,7,8,12,15,16, אך גירויים אלה יכולים להגביל ביו היישום של הידרוג&#39;ל. הידרוג-crosslinked DNA polyacrylamide (ג&#39;לים DNA) הם מצעי אלסטי דו ממדים דינמיים. crosslinks DNA מאפשר לאפנון מרחב, בזמן, והפיך של ג&#39;ל DNA נוקשות על ידי תוספת של DNA חד גדילים (ssDNA) לתקשורת או חיץ 9-11,13,14,18,21. שלא כמו ג&#39;לי הדינמי האמור שבו גירויים מוחלים לאפנון של גמישות, ג&#39;לים DNA מסתמך על דיפוזיה של ssDNA שימושי לשינוי של גמישות. לכן, המשטח העליון ג&#39;ל, שבו תאים גדלים, הוא האזור הראשון המווסת בגלל o השיעוראפנון גמישות f תלוי בעובי ג&#39;ל. ג&#39;לי DNA דומה לעמיתיהם ג&#39;ל הרשות שביש להם עמוד שדרה polyacrylamide, לעומת זאת crosslinks bis-acrylamide מוחלף בcrosslinks המורכב מדנ&quot;א (איור 1). שתי ssDNAs (SA1 וSA2) להכליא עם גדיל crosslinker (L2) כדי לפצות crosslinks DNA של ג&#39;ל. SA1 וSA2 יש רצפים שונים ששניהם מכילים שינוי Acrydite בסוף 5&#39;להתאגדות אפקטיבית לרשת של הרשות הפלסטינית. להכנת ג&#39;לי, SA1 וSA2 הם polymerized בנפרד לתוך עמוד השדרה של הרשות הפלסטינית ו, לאחר מכן, SA1 וSA2 polymerized מעורבבים יחד. L2, crosslinker, מתווסף לSA1 ותערובת SA2. רצף בסיס L2 הוא משלים לשני רצפי SA1 וSA2 וL2 hybridizes עם SA1 בתוספת SA2 כדי ליצור את crosslinks DNA. גמישות ראשונית, ג&#39;ל DNA נקבעה על ידי שני ריכוזי L2 וcrosslinking (לוחות 1 </strong&gt; ו2). ג&#39;לי DNA המכיל כמויות stoichiometric שווה של L2, SA1, וSA2 הוא ג&#39;לי הנוקש בגלל SA1 וSA2 100% crosslinked ידי L2 (מיועד כג&#39;לי 100%). ריכוזים נמוכים של תוצאת L2 בשיעור נמוך יותר של crosslinking DNA, ולכן, ג&#39;לים DNA רך יותר. ג&#39;לים נמוך כמו 50% crosslinked (מיועד כג&#39;לי 50%) נבנה 9-11. <img alt="איור 1" src="/files/ftp_upload/51323/51323fig1highres.jpg" /> איור 1 crosslinking ג&#39;ל DNA ו9-11,13,14,18,21 סכמטי uncrosslinking שלב 1:. SA1 (אדום) וSA2 (כחול) הם polymerized בנפרד לתוך עמוד השדרה polyacrylamide (שחור). לאחר פילמור, פתרונות polymerized SA1 וSA2 מעורבבים יחד. שלב 2: L2 (ירוק) הוא הוסיף וhybridizes עם SA1 בתוספת SA2 כדי ליצור את crosslinks של ג&#39;ל. שלב 3: R2 hybridizes עם tהוא דריסת הרגל של L2. שלב 4: הכלאת דריסת רגל של R2 מניעה את הרוכסן של L2 מSA1 וSA2. שלא כמו ג&#39;לי הרשות הפלסטינית, ג&#39;לים DNA יכול להקשיח ולרכך לאחר סינתזה. מסיבה זו, תאים שגודל על ג&#39;לי DNA יכולים להיות נתונה לשינויים קשיחות דינמיות. כדי להקשיח ג&#39;לי תא חסיד, ניתן להוסיף L2 לתקשורת והתרבות של ג&#39;לים אחוז הנמוך כדי להגדיל את האחוז של crosslinks. כדי לרכך ג&#39;לי תא חסיד, ניתן להסיר L2 כדי להקטין את אחוז crosslinks 10,13,21. יש L2 רצף דריסת רגל נוסף בסוף 3&#39;לאפשר L2 לuncrosslink מSA1 וSA2 (טבלת 1). הסרת L2 מושגת על ידי הכלאה של גדיל היפוך נקרא R2. R2 הוא משלים לכל אורכו של L2 וhybridizes ראשון עם דריסת רגל L2. הכלאה דריסת רגל מניעה את הרוכסן של L2 מSA1 וSA2, אשר מבטל את crosslink ומפחית את נוקשות ג&#39;ל. בדוח זה ו<html> וידאו, צעד אחר צעד הוראות ניתנות להכנת התקשות וריכוך ג&#39;לי DNA. בעוד הכנות ג&#39;ל 100% ו80% ​​מתוארים, פרוטוקול זה יכול להיות מותאם כדי ליצור ג&#39;לים DNA של אחוזי crosslinked הראשוניים וסופיים אחרים. באופן כללי, ג&#39;לי 100% ו80% ​​מוכנים, משותק על תלושי כיסוי זכוכית, פונקציונליות, וזרע עם תאים. L2 מתווסף לתקשורת של 80 ג&#39;לי% וR2 מתווסף לתקשורת של 100 ג&#39;לי%, 48 שעות לאחר ציפוי. התוספת של L2 לתקשורת מקשיחה 80 ג&#39;לי% ל100% crosslinked, ואילו התוספת של R2 לתקשורת מרככת 100 ג&#39;לי% 80% crosslinked. ג&#39;לים התקשח יועדו כ80 → 100 ג&#39;לי% וג&#39;לים התרכך יועדו כ100 → 80 ג&#39;לי% בטקסט. לשליטה או ג&#39;לים סטטי, ssDNA בהיקף של Ts או כפי שנמסר למערכת נוספת של 100% ו80 ג&#39;לי%. לאחר מינימום של שני ימים הבאים אפנון גמישות, יכולים להיות מעובד תאים ונותחו. <table border="0" cellpadding="0" cellspacing="0" fo:k</html style=";text-align:right;direction:rtl">יפ-together.within-page = &quot;תמיד&quot;&gt; <tbody align="center"><tr><td rowspan="2"></td><td rowspan="2"> crosslink DNA </td><td rowspan="2"> # של בסיסים </td><td> רצף (דריסת רגל) </td><td rowspan="2"> שינוי </td><td rowspan="2"> (T מ &#39;, ° C) טמפרטורת התכה </td><td rowspan="2"> תגובות </td></tr><tr><td> 5 &#39;→ 3&#39; </td></tr><tr><td rowspan="4"> עיצוב 1 </td><td> SA1 </td><td> 10 </td><td> GCA CCT TTG C </td><td> 5 &#39;Acrydite </td><td> 34.9 </td><td></td></tr><tr><td> SA2 </td><td> 10 </td><td> GTC AGA ATG </td><td> 5 &#39;Acrydite </td><td> 23.6 </td><td></td></tr><tr><td> L2 </td><td> 30 </td><td> TCA TTC TGA C GC AAA GGT GC G CTA CAC TTG </td><td></td><td> 56 </td><td>רצף דריסת רגל 10 נ&quot;ב כלול. </td></tr><tr><td> R2 </td><td> 30 </td><td> TAG CAA GTG CGC ACC TTT gcg TCA GAA TGA </td><td></td><td></td><td> R2 הוא משלים לL2 </td></tr><tr><td rowspan="4"> עיצוב 2 </td><td> SA1 </td><td> 14 </td><td> CGT GGC ATA GGA CT </td><td> 5 &#39;Acrydite </td><td> 46.9 </td><td></td></tr><tr><td> SA2 </td><td> 14 </td><td> GTT TCC CAA TCA GA </td><td> 5 &#39;Acrydite </td><td> 40.2 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCT GAT TGG GAA AC GTC CTA TGC CAC G GT TAC CTT CAT C </td><td></td><td> 65.9 </td><td> דריסת רגל רצף נ&quot;ב 12 כלולה. </td></tr><tr><td> R2 </td><td> 40 </td><td> GAT GAA GGT AAC CGT GGC ATA GGA CTG TTT CCC AAT CAG </td><td></td><td> 65.9 </td><td> R2 הוא משלים לL2 </td></tr><tr><td rowspan="3"> Design 3 </td><td> SA1 </td><td> 20 </td><td> ACG GAG GTG TAT GCA ATG TC </td><td> 5 &#39;Acrydite </td><td> 55 </td><td></td></tr><tr><td> SA2 </td><td> 20 </td><td> TAG GCT CAT GGA CGA CTG GA </td><td> 5 &#39;Acrydite </td><td> 56.6 </td><td></td></tr><tr><td> L2 </td><td> 40 </td><td> TCC AGT CGT CCC TAA GCA TG G ACA TTG CAT ACA CCT CCG T </td><td></td><td> 68.8 </td><td> דריסת הרגל אינה כלולה. </td></tr><tr><td rowspan="2"> בקרה </td><td rowspan="2"> בקרה </td><td rowspan="2"> 20-40 </td><td> AAA AAA (וכו &#39;) או </td><td rowspan="2"></td><td rowspan="2"></td><td rowspan="2"></td></tr><tr><td> TTT TTT (וכו &#39;) </td></tr></tbody></table> .1 רצפי בסיס השולחן לssDNA 9-11,13,14,18,21. נייד ומחקרים מכאניים נצלו כמה דעיצובי crosslink ifferent ליצור ג&#39;לי DNA עם מגוון רחב של תכונות מכאניות סטטי ודינמיות. הפרמטרים מווסת בעיצוב crosslink הם רצף בסיס ואורך רצף או אורך crosslink. גופנים מודגש ונטויים להמחיש זיווג בסיס בין SA1 וL2 ובין SA2 וL2, בהתאמה. <table border="0" cellpadding="0" cellspacing="0" fo:keep-together.within-page="always" style=";text-align:right;direction:rtl"><tbody align="center"><tr><td rowspan="2"></td><td colspan="8"> עיצוב </td></tr><tr><td colspan="3"> 1 </td><td colspan="3"> 2 </td><td colspan="2"> 3 </td></tr><tr><td> ריכוז acrylamide (%) </td><td colspan="3"> 10 </td><td colspan="3"> 10 </td><td> 10 </td><td> 4 </td></tr><tr><td> SA1 בתוספת SA2 הכלאה לL2 (% crosslinked) </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 50 </td><td> 80 </td><td> 100 </td><td> 100 </td><td> 100 </td></tr><tr><td> &lt;strong&gt; אלסטיות (kPa, ממוצע ± SEM) </td><td> 6.6 ± 0.6 </td><td> 17.1 ± 0.8 </td><td> 29.8 ± 2.5 </td><td> 5.85 ± 0.62 </td><td> 12.67 ± 1.33 </td><td> 22.88 ± 2.77 </td><td> 25.2 ± 0.5 </td><td> 10.4 ± 0.6 </td></tr></tbody></table> לוח 2: מודול יאנג (E) של ג&#39;לים DNA 9-11,13,14,18,21. ריכוז Acrylamide, אחוז crosslink, ואורך crosslink יכול להיות מווסת בג&#39;לים DNA. יש עיצובים 1, 2, ו -3 20, 28, ו40 אורכי crosslink נ&quot;ב, בהתאמה. יש לי 100 ג&#39;לי% עבור כל העיצובים moduli דומה המצביע על אורך crosslink אינו משפיע על אלסטיות ג&#39;ל. עם זאת, שינויים בריכוז acrylamide לשנות גמישות ג&#39;ל DNA.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>ssDNA</td>
			<td>Integrated DNA Technologies (Coralville, Iowa) idtdna.com</td>
			<td></td>
			<td>Do not vortex ssDNA. Gentle invert the vial and/or pipette solution to mix.</td>
		</tr>
		<tr>
			<td>PBS with calcium and magnesium</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>100X Tris-EDTA buffer (TE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO) sigmaldrich.com</td>
			<td>T9285</td>
			<td></td>
		</tr>
		<tr>
			<td>10X Tris-Borate-EDTA buffer (TBE buffer)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>93290</td>
			<td>TBE is a reproductive toxin.</td>
		</tr>
		<tr>
			<td>40% Acrylamide solution</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>BP14021</td>
			<td>Acrylamide is a toxin.</td>
		</tr>
		<tr>
			<td>Ammonium persulfate (APS)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>A3678</td>
			<td>Prepare a 2% solution in TE buffer. APS is a toxin and irratant.</td>
		</tr>
		<tr>
			<td>Tetramethylethylenediamine (TEMED)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>T9281</td>
			<td>Prepare a 20% solution in TE buffer. TEMED is flammable, a corrosive, and a toxin.</td>
		</tr>
		<tr>
			<td rowspan="2">12-mm diameter round coverglass</td>
			<td rowspan="2">Fisher Scientific (Pittsburg, PA) fishersci.com</td>
			<td rowspan="2">12-545-82</td>
			<td rowspan="2"></td>
		</tr>
		<tr>
			<td>Norland optical adhesive 72</td>
			<td>Norland Products (Cranbury, NJ) norlandprod.com</td>
			<td>NOA72</td>
			<td></td>
		</tr>
		<tr>
			<td rowspan="2">24-well tissue culture plate</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td rowspan="2">Microcentrifuge tubes</td>
			<td rowspan="2"></td>
			<td rowspan="2"></td>
			<td rowspan="2">Any brand.</td>
		</tr>
		<tr>
			<td>Sulfo-SANPAH</td>
			<td>ProteoChem or Thermo Fisher, (Rockland, IL) proteochem.com or thermofisher.com</td>
			<td>C111 or 22589</td>
			<td>Prepare a 0.315 mg/ml solution in water immediately before use. Dissolve at 37&#176;C and filter sterilze. It is normal to observe undisolved sulfo-SANPAH in the filter. Sulfo-SANPAH is light sensitive and, therefore, the solution should be protect from light until UV exposure.</td>
		</tr>
		<tr>
			<td>Poly-D-Lysine (PDL)</td>
			<td>Sigma-Aldrich (St. Loius, MO)</td>
			<td>P6407</td>
			<td>Prepare a 0.2 mg/ml solution in water and filter sterilize.</td>
		</tr>
		<tr>
			<td>Collagen Type I</td>
			<td>Affymetrix (Santa Clara, CA) affymetrix.com</td>
			<td>13813</td>
			<td>Prepare a 0.2 mg/ml solution in 0.2 N acetic acid. Solution needs to remain cold at all times to avoid polymerization. Acetic acid is a flammable, toxic, and corrosive.</td>
		</tr>
		<tr>
			<td>22 X 60 cover glass</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>12-544-G</td>
			<td></td>
		</tr>
		<tr>
			<td>Positive-displacement pipette</td>
			<td>Gilson, Inc (Middletown, WI) gilson.com</td>
			<td>F148504</td>
			<td></td>
		</tr>
		<tr>
			<td>Heat block</td>
			<td>Fisher Scientific (Pittsburg, PA)</td>
			<td>11-718</td>
			<td></td>
		</tr>
		<tr>
			<td>UV light source</td>
			<td></td>
			<td></td>
			<td>Place gels as close as possible to the UV light. UV light can cause skin or eye injury.</td>
		</tr>
		<tr>
			<td>Thermometer</td>
			<td></td>
			<td></td>
			<td>Any brand.</td>
		</tr>
		<tr>
			<td>Nitrogen gas</td>
			<td>GTS-Welco (Flemington, NJ) www.praxairmidatlantic.com/</td>
			<td>NI 5.0UH-R</td>
			<td></td>
		</tr>
	</tbody>
</table>

preparation of dna-crosslinked polyacrylamide hydrogels

Mechanobiology הוא תחום מדעי מתפתח העוסק בתפקיד הקריטי של רמזים פיזיים בהכוונת מורפולוגיה תא ותפקוד. לדוגמא, ההשפעה של גמישות רקמות בתפקוד תא הוא אזור מרכזי של מחקר mechanobiology כי רקמות נוקשות מודולציה עם מחלה, פיתוח, ופציעה. חומרי סטטי מחקה רקמות, או חומרים שלא ניתן לשנות את קשיחות פעם תאים מצופה, המשמשים ברובה כדי לחקור את ההשפעות של רקמות נוקשות בפונקציות תא. בעוד שהמידע שנאסף ממחקרי סטטי הוא בעל ערך, מחקרים אלה אינם מעידים על האופי הדינמי של microenvironment הסלולרי in vivo. כדי לטפל טוב יותר את ההשפעות של קשיחות דינמית על תפקוד תאים, שפיתחנו מערכת crosslinked DNA הידרוג&#39;ל polyacrylamide (ג&#39;לים DNA). שלא כמו מצעים דינמיים אחרים, יש לי ג&#39;לי DNA היכולת להגדיל או להקטין בקשיחות לאחר הייצור ללא גירויים. ג&#39;לים DNA מורכב מcrossl DNAסוגי הדיו שpolymerized לתוך עמוד השדרה polyacrylamide. הוספה והסרה של crosslinks באמצעות משלוח של גדילי דנ&quot;א יחיד מאפשרת מרחב, בזמן, ושליטה הפיכה של גמישות ג&#39;ל. שהראינו בדוחות קודמים שאפנון דינמי של גמישות ג&#39;ל DNA משפיע פיברובלסטים ונוירון התנהגות. בדוח זה ווידאו, אנו מספקים סכמטי המתאר את מנגנוני crosslinking ג&#39;ל DNA והוראות צעד אחר צעד על ג&#39;לי DNA ההכנה.

לפני המחקרים שלנו, אינטראקציות התא-ECM נצפו בחומרים ביולוגיים תואמים סטטי או על מצעים דינמיים בלתי הפיכים וחד כיווני. מצעים אלו אינם משקפים את האופי הדינמי של microenvironment הסלולרי. העבודה שלנו מסיטה את פרדיגמות טכניות הקיימות על ידי מתן מודל פיסיולוגי יותר לחקר אינטראקצי...

Watch this Scientific Journal Video about Preparation of DNA-crosslinked Polyacrylamide Hydrogels at JoVE.com

Preparation of DNA-crosslinked Polyacrylamide Hydrogels

המעבדה שלנו פיתחה הידרוג-crosslinked DNA polyacrylamide, מערכת הידרוג&#39;ל דינמית, כדי להבין טוב יותר את ההשפעות של ויסות רקמות נוקשות על תפקוד תא. כאן, אנו מספקים שרטוטים, תיאור, ופרוטוקולים להכנת הידרוג אלה.

הכנת-crosslinked DNA polyacrylamide הידרוג

Mechanobiology is an emerging scientific area that addresses the critical role of physical cues in directing cell morphology and function. For example, ...

preparation-of-dna-crosslinked-polyacrylamide-hydrogels

Research

JoVE Journal

Biology

14.4K Views.  JFK Medical Center.  המעבדה שלנו פיתחה הידרוג-crosslinked DNA polyacrylamide, מערכת הידרוג'ל דינמית, כדי להבין טוב יותר את ההשפעות של ויסות רקמות נוקשות על תפקוד תא. כאן, אנו מספקים שרטוטים, תיאור, ופרוטוקולים להכנת הידרוג אלה. 

Video: Preparation of DNA-crosslinked Polyacrylamide Hydrogels

Fast and Sensitive Colloidal Coomassie G-250 Staining for Proteins in Polyacrylamide Gels

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and high quantitation. Furthermore, it is completely compatible with mass spectrometric protein identification. But despite these advantages, CBB is regarded to be less sensitive than silver or fluorescence stainings and therefore rarely used for the detection of proteins in analytical gel-based proteomic approaches.
Several improvements of the original Coomassie protocol1 have been made to increase the sensitivity of CBB. Two major modifications were introduced to enhance the detection of low-abundant proteins by converting the dye molecules into colloidal particles: In 1988, Neuhoff and colleagues applied 20% methanol and higher concentrations of ammonium sulfate into the CBB G-250 based staining solution2, and in 2004 Candiano et al. established Blue Silver using CBB G-250 with phosphoric acid in the presence of ammonium sulfate and methanol3. Nevertheless, all these modifications just allow a detection of approximately 10 ng protein. A widely fameless protocol for colloidal Coomassie staining was published by Kang et al. in 2002 where they modified Neuhoff's colloidal CBB staining protocol regarding the complexing substances. Instead of ammonium sulfate they used aluminum sulfate and methanol was replaced by the less toxic ethanol4. The novel aluminum-based staining in Kang's study showed superior sensitivity that detects as low as 1 ng/band (phosphorylase b) with little sensitivity variation depending on proteins.
Here, we demonstrate application of Kang's protocol for fast and sensitive colloidal Coomassie staining of proteins in analytical purposes. We will illustrate the quick and easy protocol using two-dimensional gels routinely performed in our working group.

This video shall popularize a colloidal Coomassie G-250 staining protocol according to Kang et al. for the detection of average 4 ng protein in gels. The staining is completed within 2 hours and without any effort. We routinely use Kang's protocol for analytical purposes in gel-based proteomics.

מהיר רגיש Colloidal Coomassie G-250 מכתים עבור חלבונים ג&#39;ל polyacrylamide

Coomassie Brilliant Blue (CBB) is a dye commonly used for the visualization of proteins separated by SDS-PAGE, offering a simple staining procedure and ...

Denaturing Urea Polyacrylamide Gel Electrophoresis (Urea PAGE)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their separation in a polyacrylamide gel matrix based on the molecular weight. Fragments between 2 to 500 bases, with length differences as small as a single nucleotide, can be separated using this method1. The migration of the sample is dependent on the chosen acrylamide concentration. A higher percentage of polyacrylamide resolves lower molecular weight fragments. The combination of urea and temperatures of 45-55 &deg;C during the gel run allows for the separation of unstructured DNA or RNA molecules.
In general this method is required to analyze or purify single stranded DNA or RNA fragments, such as synthesized or labeled oligonucleotides or products from enzymatic cleavage reactions.
In this video article we show how to prepare and run the denaturing urea polyacrylamide gels. Technical tips are included, in addition to the original protocol 1,2.

Denaturing Urea Polyacrylamide Gel Electrophoresis Urea PAGE

Denaturing urea polyacrylamide gel electrophoresis is used to separate single-stranded DNA or RNA up to a limit of 500 nucleotides. Urea in combination with heat denatures samples and unstructured single strands migrate within the gel matrix according to their molecular weight.

Denaturing polyacrylamide דשן ג&#39;ל אלקטרופורזה (עמוד דשן)

Urea PAGE or denaturing urea polyacrylamide gel electrophoresis employs 6-8 M urea, which denatures secondary DNA or RNA structures and is used for their ...

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

Encapsulation נייד ב הידרוג 3D עבור הנדסת רקמות

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

Blue Native Polyacrylamide Gel Electrophoresis (BN-PAGE) for Analysis of Multiprotein Complexes from Cellular Lysates

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other proteins (Sali, Glaeser et al. 2003). Thus, the study of protein-protein interaction networks requires the detailed characterization of MPCs to gain an integrative understanding of protein function and regulation. For identification and analysis, MPCs must be separated under native conditions. In this video, we describe the analysis of MPCs by blue native polyacrylamide gel electrophoresis (BN-PAGE). BN-PAGE is a technique that allows separation of MPCs in a native conformation with a higher resolution than offered by gel filtration or sucrose density ultracentrifugation, and is therefore useful to determine MPC size, composition, and relative abundance (Sch&auml;gger and von Jagow 1991); (Sch&auml;gger, Cramer et al. 1994). By this method, proteins are separated according to their hydrodynamic size and shape in a polyacrylamide matrix. Here, we demonstrate the analysis of MPCs of total cellular lysates, pointing out that lysate dialysis is the crucial step to make BN-PAGE applicable to these biological samples. Using a combination of first dimension BN- and second dimension SDS-PAGE, we show that MPCs separated by BN-PAGE can be further subdivided into their individual constituents by SDS-PAGE. Visualization of the MPC components upon gel separation is performed by standard immunoblotting. As an example for MPC analysis by BN-PAGE, we chose the well-characterized eukaryotic 19S, 20S, and 26S proteasomes.

Blue Native Polyacrylamide Gel Electrophoresis BN-PAGE for Analysis of Multiprotein Complexes from Cellular Lysates

In this video, we describe the characterization of multiprotein complexes (MPCs) by blue native polyacrylamide gel electrophoresis (BN-PAGE). In a first dimension, dialyzed cellular lysates are separated by BN-PAGE to identify individual MPCs. In a second dimension SDS-PAGE, MPCs of interest are further subdivided to analyze their constituents by immunoblotting.

ג&#39;ל כחול Native polyacrylamide אלקטרופורזה (BN-PAGE) עבור ניתוח של מתחמי multiprotein מ lysates נייד

Multiprotein complexes (MPCs) play a crucial role in cell signalling, since most proteins can be found in functional or regulatory complexes with other ...

Mechanical Stimulation of Chondrocyte-agarose Hydrogels

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy this deficiency, several medical interventions have been developed. One such method is to resurface the damaged area with tissue-engineered cartilage; however, the engineered tissue typically lacks the biochemical properties and durability of native cartilage, questioning its long-term survivability. This limits the application of cartilage tissue engineering to the repair of small focal defects, relying on the surrounding tissue to protect the implanted material. To improve the properties of the developed tissue, mechanical stimulation is a popular method utilized to enhance the synthesis of cartilaginous extracellular matrix as well as the resultant mechanical properties of the engineered tissue. Mechanical stimulation applies forces to the tissue constructs analogous to those experienced in vivo. This is based on the premise that the mechanical environment, in part, regulates the development and maintenance of native tissue1,2. The most commonly applied form of mechanical stimulation in cartilage tissue engineering is dynamic compression at physiologic strains of approximately 5-20% at a frequency of 1 Hz1,3. Several studies have investigated the effects of dynamic compression and have shown it to have a positive effect on chondrocyte metabolism and biosynthesis, ultimately affecting the functional properties of the developed tissue4-8. In this paper, we illustrate the method to mechanically stimulate chondrocyte-agarose hydrogel constructs under dynamic compression and analyze changes in biosynthesis through biochemical and radioisotope assays. This method can also be readily modified to assess any potentially induced changes in cellular response as a result of mechanical stimuli.

The biosynthesis of cartilaginous extracellular matrix by chondrocytes can be affected by application of mechanical stimuli. This method describes the technique of applying dynamic compressive strains to chondrocytes encapsulated in 3D constructs and the evaluation of induced changes in chondrocyte metabolism.

גירוי מכאני של הידרוג Chondrocyte-agarose

Articular cartilage suffers from a limited repair capacity when damaged by mechanical insult or degraded by disease, such as osteoarthritis. To remedy ...

FRET Imaging in Three-dimensional Hydrogels

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly employ two-dimensional (2D) culture, which does not mimic the three-dimensional (3D) cellular microenvironment. A method to perform quenched emission FRET imaging using conventional widefield epifluorescence microscopy of cells within a 3D hydrogel environment is presented. Here an analysis method for ratiometric FRET probes that yields linear ratios over the probe activation range is described. Measurement of intracellular cyclic adenosine monophosphate (cAMP) levels is demonstrated in chondrocytes under forskolin stimulation using a probe for EPAC1 activation (ICUE1) and the ability to detect differences in cAMP signaling dependent on hydrogel material type, herein a photocrosslinking hydrogel (PC-gel, polyethylene glycol dimethacrylate) and a thermoresponsive hydrogel (TR-gel). Compared with 2D FRET methods, this method requires little additional work. Laboratories already utilizing FRET imaging in 2D can easily adopt this method to perform cellular studies in a 3D microenvironment. It can further be applied to high throughput drug screening in engineered 3D microtissues. Additionally, it is compatible with other forms of FRET imaging, such as anisotropy measurement and fluorescence lifetime imaging (FLIM), and with advanced microscopy platforms using confocal, pulsed, or modulated illumination.

F&#246;rster resonance energy transfer (FRET) imaging is a powerful tool for real-time cell biology studies. Here a method for FRET imaging cells in physiologic three-dimensional (3D) hydrogel microenvironments using conventional epifluorescence microscopy is presented. An analysis for ratiometric FRET probes that yields linear ratios over the activation range is described.

סריג הדמיה ב הידרוג תלת ממדי

Imaging of F&#246;rster resonance energy transfer (FRET)&#160;is a powerful tool for examining cell biology in real-time. Studies utilizing FRET commonly ...

Proteolytically Degraded Alginate Hydrogels and Hydrophobic Microbioreactors for Porcine Oocyte Encapsulation

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of assisted reproduction techniques such as oocyte in vitro maturation (IVM), in vitro fertilization (IVF) and cloning of domestic animals by nuclear transfer from somatic cell. IVM is the method particularly of significance. It is the platform technology for the supply of mature, good quality oocytes for applications such as reduction of the generation interval in commercially important or endangered species, research concerning in vitro human reproduction, and production of transgenic animals for cell therapies. The term oocyte quality includes its competence to complete maturation, be fertilized, thereby resulting in healthy offspring. This means that oocytes of good quality are paramount for successful fertilization including IVF procedures. This poses many difficulties to develop a reliable culture method that would support growth not only of human oocytes but also of other large mammalian species. The first step in IVM is the in vitro culture of oocytes. This work describes two protocols for the 3D culture of porcine oocytes. In the first, 3D model cumulus-oocyte complexes (COCs) are encapsulated in a fibrin-alginate bead interpenetrating network, in which a mixture of fibrin and alginate are gelled simultaneously. In the second one, COCs are suspended in a drop of medium and encapsulated with fluorinated ethylene propylene (FEP; a copolymer of hexafluoropropylene and tetrafluoroethylene) powder particles to form microbioreactors defined as Liquid Marbles (LM). Both 3D systems maintain the gaseous in vitro culture environment. They also maintain COCs 3D organization by preventing their flattening and consequent disruption of gap junctions, thereby preserving the functional relationship between the oocyte, and surrounding follicular cells.

Presented here are two protocols for the encapsulation of porcine oocytes in 3D culture conditions. In the first, cumulus-oocyte complexes (COCs) are encapsulated in fibrin-alginate beads. In the second, they are enclosed with fluorinated ethylene propylene powder particles (microbioreactors). Both systems ensure optimal conditions to maintain their 3D organization.

הידרוג'לים אלג'ינאט מפורקים ופרוטאוליטית ומיקרוביו-סיבה לאנקלוסציה של פורצין אוציט

In reproductive biology, the biotechnology revolution that began with artificial insemination and embryo transfer technology led to the development of ...

Detection of Glycosaminoglycans by Polyacrylamide Gel Electrophoresis and Silver Staining

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role in a variety of basic biological structures and processes. As polymers, GAGs exist as a polydisperse mixture containing polysaccharide chains that can range from 4000 Da to well over 40,000 Da. Within these chains exists domains of sulfation, conferring a pattern of negative charge that facilitates interaction with positively charged residues of cognate protein ligands. Sulfated domains of GAGs must be of sufficient length to allow for these electrostatic interactions. To understand the function of GAGs in biological tissues, the investigator must be able to isolate, purify, and measure the size of GAGs. This report describes a practical and versatile polyacrylamide gel electrophoresis-based technique that can be leveraged to resolve relatively small differences in size between GAGs isolated from a variety of biological tissue types.

This report describes techniques to isolate and purify sulfated glycosaminoglycans (GAGs) from biological samples and a polyacrylamide gel electrophoresis approach to approximate their size. GAGs contribute to tissue structure and influence signaling processes via electrostatic interaction with proteins. GAG polymer length contributes to their binding affinity for cognate ligands.

זיהוי של גליקוסמינוגליקנס על ידי אלקטרופורזה ג'ל פוליאקרילמיד וכתמים כסף

Sulfated glycosaminoglycans (GAGs) such as heparan sulfate (HS) and chondroitin sulfate (CS) are ubiquitous in living organisms and play a critical role ...

An Improved Chemotaxis Assay for the Rapid Identification of Rhizobacterial Chemoattractants in Root Exudates

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward method to quickly identify the chemoattractants that could induce the chemotactic movement of rhizosphere growth-promoting bacteria on sterile glass slides via simple steps. Bacteria solution (OD600 = 0.5) and sterile chemoattractant aqueous solution were added dropwise on the glass slide at an interval of 1 cm. An inoculating loop was used to connect the chemoattractant aqueous solution to the bacterial solution. The slide was kept at room temperature for 20 min on the clean bench. Finally, the chemoattractant aqueous solution was collected for bacterial counting and microscopic observation. In this study, through multiple comparisons of experimental results, the method overcame multiple shortcomings of traditional bacterial chemotaxis methods. The method reduced the error of plate counting and shortened the experimental cycle. For the identification of chemoattractant substances, this new method can save 2-3 days compared to the traditional method. Additionally, this method allows any researcher to systematically complete a bacterial chemotaxis experiment within 1-2 days. The protocol can be considered a valuable resource for understanding plant-microbe interactions.

Here, we present an improved chemotaxis assay protocol. The goal of this protocol is to reduce the steps and costs of traditional bacterial chemotaxis methods and to serve as a valuable resource for understanding plant-microbe interactions.

בדיקת כימוטקסיס משופרת לזיהוי מהיר של כימותרפים ריזובקטריאליים באקסודות שורש

Chemotaxis identification is very important for the research and application of rhizosphere growth-promoting bacteria. We established a straightforward ...

הטמעת תגובות CFPS במגוון מטריצות הידרוג'ל

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

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

מחבר זרקור: גישה חדשנית להטמעת תגובות סינתזת חלבונים ללא תאים בהידרוג'לים 

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

שיטות להטמעת תגובות סינתזת חלבונים ללא תאים בהידרוג'לים בקנה מידה מאקרו

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