Thomas Bell, Lyn Mattathil, Mark Mannino ve Neej Patel ve teknik destek için teşekkür etmek istiyorum. Bu eser Jonathan Fisher için Amerika Birleşik Devletleri halk sağlığı hizmet Ödülü R15DK102122 diyabet Ulusal Enstitüsü ve sindirim ve böbrek hastalıkları (NIDDK) tarafından desteklenmiştir. El yazması içerik sadece yazarlar sorumludur ve mutlaka NIDDK veya Ulusal Sağlık Enstitüleri resmi görüşlerini temsil etmiyor.

Not: Resim 1 anahtar adımlar şematik bir bakış için bkz:.
1. WST-1 azaltma tahlil
<ol><li>Standart hücre kültür yordamlar7 satır A-f kullanan bir 96-şey tabak içinde kullanarak C2C12 yapışık hücreleri ayırt etmek ve büyümek<ol>
<li>İn Dulbecco'nın modifiye kartal orta (%2 at serum, 100 U/mL penisilin ve 0.1 mg/mL streptomisin ile desteklenmiş DMEM) oluşan bir farklılaşma aracı kullanın. %5 CO237 ° C'de hücreler kuluçkaya.</li>
		<li>TPMET askorbat katılımı izlerken, farklılaşma medya 100 µM askorbik asit ile ek. Hücre farklılaşması medya ~ 24-48 için kuluçkaya izin h.</li>
	</ol>
</li>
	<li>Hisse senedi WST-1 ve PMS çözümleri hazırlayın.<ol>
<li>Bir 10 mM hisse senedi WST-1 yapmak için WST-1 0,033 g dağıtılması (formül ağırlık (FW): 651.34 g/mol) 5 mL fosfat tamponlu tuz (PBS). 4 ° C'de mağaza</li>
		<li>PMS 5 mM stokunun yapmak, PMS 0.0023 g dağıtılması (FW: 306.34 g/mol) 1,5 ml deiyonize H2O (diH2O). -20 ° C'de depolayın ve ışıktan korumak.</li>
	</ol>
</li>
	<li>İçin PBS veya HEPES 11,4 ml 5 mM son bir konsantrasyon arabelleğe alınmış serum glikoz 0.0108 g ekleyin (HBS; 20 mM HEPES sodyum tuz, 140 mM NaCl, 5 mM KCl, 2.5 mM MgSO4, 1 mM CaCl2). 10 mm WST-1 400 µM son bir konsantrasyon ve 5 mm PMS 20 µM son bir konsantrasyon için 48 µL için 480 µL ekleyin.</li>
	<li>TPMET askorbat katılımı izlerken, çözüm iki 6 mL aliquots bölün. Bir aliquot için Ekle diH2O 6 µL ve diğer aliquot olarak 2 kU/mL AO 2 U/mL nihai bir konsantrasyon için 6 µL ekleyin.</li>
	<li>TPMET katılımı süperoksit izlerken, çözüm iki 6 mL aliquots bölün. Bir aliquot için 0,1 M KPO4 arabelleği 55 µL ekleyin ve diğer aliquot 6.5 kU/ml SOD 60 U/mL nihai bir konsantrasyon için 55 µL ekleyin.</li>
	<li>PBS hücrelerle yıkayın. Medya Aspire edin ve 150 µL PBS ekleyin. O zaman PBS Aspire edin. Not: Kaplama, ekli hücrelerle tahlil yapılır. Böylece, hiçbir aralıklarla veya tripsin yıkama işleminin kullanımı olduğunu.</li>
	<li>WST-1 çözüm (-) 100 µL eklemek SOD veya (-) AO sütunlara 1-6 ve 100 µL WST-1 çözüm (+) SOD veya (+) AO 96-şey plaka 7-12 sütuna ekleyin. G ve H satır arka plan denetimleri kullanın (değişikliği izlemek içinYani, Wells hücreleri olmadan tek başına reaktif Absorbans içinde).</li>
	<li>438, 1 h için her 10 min Spektrofotometre kullanarak Absorbans değerleri ölçmek nm.</li>
	<li>Okuma sonra medya Aspire edin ve her yıkama PBS 150 µL ile iyi. O zaman PBS Aspire edin.</li>
	<li>Her şey için Absorbans değişimi Absorbans için o kadar iyi herhangi bir zamanda noktada bir kuyudan için ilk Absorbans çıkararak hesaplar. Bu değişiklik arka plan wells (Yani, wells ile tahlil çözüm ama hiçbir hücre) gözlenen Absorbans (varsa) yapılan değişikliğin düzeltin.</li>
	<li>Analiz için 60 dk kontrol ölçüm Absorbans verileri normalleştirmek veya wells PBS veya HBS ile yıkayın ve bir bicinchoninic asit (BCA) protein tahlil gerçekleştirin.</li>
	<li>2 mg/mL sığır serum albumin (BSA) standart Ekle (aralığı 0.5-2 hücreleri izdiham derecesine bağlı olarak standart Curve µL) boş wells için (satır G ve H), sonra BCA reaktif için bütün wells ekleyin..</li>
	<li>Nmol protein µg başına WST-1 azaltılması yoluyla veri ölçmek. 37 mM-1 cm-1 22 tükenme katsayısı azaltılmış WST-1'de 438 kullanmak nm.</li>
</ol>2. DPIP azaltma tahlil
<ol><li>Büyümek ve C2C12 yapışık hücreleri ile aynı prosedürü adım 1.1 olarak ayırt etmek.</li>
	<li>Hisse senedi 10 mM DPIP çözüm aşağıdaki gibi hazırlayın. DPIP 0,029 g dağıtılması (FW: 290.08 g/mol) diH2O. Onayla 600 Absorbans ölçme tarafından DPIP konsantrasyonu 10 ml nm bir Spektrofotometre ile. 1 mM-1 cm-1 23 tükenme katsayısı azaltılmış DPIP 600 kullanmak nm. 4 ° C'de mağaza</li>
	<li>0.0108 g glikoz PBS 11.880 mL 5 mM son bir konsantrasyon için ekleyin. 10 mm DPIP 100 µM son bir konsantrasyon için 120 µL ekleyin.</li>
	<li>TPMET askorbat katılımı izlerken, çözüm iki 6 mL aliquots bölün. Bir aliquot için Ekle diH2O 6 µL ve diğer aliquot olarak 2 kU/mL AO 2 U/mL nihai bir konsantrasyon için 6 µL ekleyin.</li>
	<li>TPMET katılımı süperoksit izlerken, çözüm iki 6 mL aliquots bölün. Bir aliquot için 0,1 M KPO4 arabelleği 55 µL ekleyin ve diğer aliquot 6.5 kU/ml SOD 60 U/mL nihai bir konsantrasyon için 55 µL ekleyin.</li>
	<li>Medya Aspire edin ve PBS 150 µL hücrelerde yıkayın. PBS Aspire edin ve DPIP çözüm (-) 100 µL eklemek SOD veya (-) AO sütunlara 1-6 ve 100 µL DPIP çözüm (+) SOD veya (+) AO 96-şey plaka 7-12 sütuna ekleyin. Arka plan denetimleri satır G ve H kullanarak (değişikliği izlemek içinYani, Wells hücreleri olmadan tek başına reaktif Absorbans içinde).</li>
	<li>600 nm kullanarak Absorbans ölçmek Spektrofotometre her 10 min için 1 h Absorbans 60 dk adımlara 1.9-1,13 benzer denetime göre değişiklik ölçmek.</li>
</ol>3. düşük elektron alıcısı yüzeylerde AO veya SOD için olup belirlenmesi
<ol><li>PBS veya HBS 5.436 mL için 10 mm WST-1 400 µM son bir konsantrasyon ve 5 mm PMS 20 µM son bir konsantrasyon için 24 µL için 240 µL ekleyin.<ol>
<li>DPIP için 10 mm DPIP, 100 µM, son bir konsantrasyon için 60 µL PBS veya HBS için 5.940 mL ekleyin.</li>
	</ol>
</li>
	<li>Her bir düz-alt 96-şey plaka yokluğunda hücreleri, iyi ve 438, Spektrofotometre Absorbans ölçmek için çözüm 100 µL eklemek için DPIP için WST-1 veya 600 nm nm.</li>
	<li>10 mM askorbat 1 µL kuyular için 100 mikron son bir konsantrasyon yarısını ekleyin. Onu stabilize kadar Absorbans izlemek.</li>
	<li>İstikrar, üzerine 200 U/mL AO 1 µL 2 U/mL nihai bir konsantrasyon için her şey için veya 60 U/mL her son bir konsantrasyon iyi ve 1 h Absorbans izlemek için 6 kU/ml SOD 1 µL ekleyebilirsiniz.</li>
</ol>

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G., Babcock, G. T., Dye, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Properties+of+5-methylphenazinium+methyl+sulfate.+Reaction+of+the+oxidized+form+with+NADH+and+of+the+reduced+form+with+oxygen.">Properties of 5-methylphenazinium methyl sulfate. Reaction of the oxidized form with NADH and of the reduced form with oxygen.</a> J Biol Chem. 257 (3), 1458-1461 (1982).</li><li>Maghzal, G. J., Krause, K. H., Stocker, R., Jaquet, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+of+reactive+oxygen+species+derived+from+the+family+of+NOX+NADPH+oxidases.">Detection of reactive oxygen species derived from the family of NOX NADPH oxidases.</a> Free Radic Biol Med. 53 (10), 1903-1918 (2012).</li></ol>

Yazarlar ifşa gerek yok.

Biz hücre dışı elektron alıcısı, WST-1 ve DPIP, C2C12 myotubes tPMET izlemek için spektrofotometrik deneyleri içinde kullanmak için iki yöntem sundu. Spektrofotometre plaka okuyucu ve hücre hatlarında standart kültür yordamlar büyüme ile tPMET bu elektron alıcısı basit Mikroplaka tahlil ile izlemek mümkündür. İyi iyi bir tahlil içinde WST-1 azaltma tekrarlanabilir değil ama gün-gün değişkenlik var. PBS arabellek olarak kullanan varyasyon (CV) günlük fark 0.18 ve arabellek 0,23 olduğu gibi...

Saf plazma membran elektron alıcısı azaltma becerisini plazma zarı doğasında Redoks kapasitesi1sahip görünümüne yol açmıştır. Daha önce görüldü mantar, bitki ve hayvan, tPMET bir işlemi birden çok organizmalar2,3,4,5' e ortak var. Saccharomyces cerevisiae, havuç hücreleri, eritrositler, lenfositler, osteosarkom, Melanom, makrofajlar, iskelet kası ve nötrofil2,3, bu işlem özellikle gösterilmiştir 4 , 5 , 6 , 7. hücre dışı oksidanlar azaltmak için plazma zarı arasında elektron taşıma bir süreç içinde tPMET de dahil olmak üzere birçok hücresel işlevler ilgilenmektedir: hücre büyüme5,8, hücre canlılığı9, demir metabolizma10,11,12,13ve Reaktif oksijen türleri12,14,15koruma sinyalizasyon hücre. Pek çok hücresel fonksiyon tPMET'ın katılımı nedeniyle, tPMET bir dengesizlik kanser16, kardiyovasküler hastalık17, dahil olmak üzere bazı ciddi sağlık koşulları, gelişimine katkıda bulunmak olan ve metabolik Sendromu18.
Plazma zarı arasında elektron transferi izlemek için birden çok yolu vardır, ancak en çok kullanılan teknik ekstraselüler elektron alıcısı Renkölçer deneyleri ile azalma değerlendirmek için. Yaygın olarak kullanılan hücre dışı elektron alıcısı tetrazolium tuzları, DPIP, FeCN ve ferricytochrome c19,20dir. En sık kullanılan tetrazolium WST-119bilinen ikinci nesil bir tuz tuzudur. Bu bileşik Renkölçer deneyleri ilk nesil tetrazolium tuzlar, su çözünürlük21artırmak iki Sülfonat grup nedeniyle göre kullanmak kolaydır. WST-1, iki tek-elektron transferi olaylar ara elektron alıcısı 1-metoksi-phenazine methosulfate (MPM), ile birlikte azalır. Bu azalma WST-1 zayıf renkli oksitlenmiş şeklinde bir daha yoğun, sarı formazan20,22' ye değişir. WST-1 37 x 103 M-1cm-1, yüksek molar tükenme katsayısı bir yüksek tahlil duyarlılık21,22' ye lider vardır. DPIP da tPMET izlemek için bir hücre dışı elektron alıcısı olarak kullanılmaktadır. Bu DPIP extracellularly tPMET ara elektron alıcısı23,24yardımı olmadan tarafından azaltılabilir gösterilmiştir. Ara elektron alıcısı olmaması nedeniyle DPIP doğrudan WST-124aksine plazma zarı pick-up elektron olabilir. Benzer şekilde DPIP, extracellularly ferrocyanide için tPMET ara elektron alıcısı19,24yardımı olmadan düşülmesi için FeCN gösterilmiştir. WST-1 ve DPIP, FeCN bir alt tahlil duyarlılık9için önde gelen bir düşük molar tükenme katsayısı vardır. TPMET izlemek için başka bir sık kullanılan hücre dışı elektron alıcısı ferricytochrome c. WST-1, ferricytochrome c azaltma artar ara elektron alıcısı, MPM22kullanımı ile benzer olduğunu. WST-1 olsa da, ferricytochrome c yöntemi yüksek bir arka plan ve düşük molar tükenme katsayısı22nedeniyle daha az duyarlıdır.
Burada tPMET spektrofotometrik deneyleri aracılığıyla gerçek zamanlı analiz için bir yöntem mevcut. Ekstraselüler elektron alıcısı ferricytochrome c gibi diğer yaygın olarak karşılaştırıldığında olmak daha ucuz kullanılan iken her ikisi de yüksek molar tükenme katsayısı gibi WST-1 ve DPIP, hücre dışı elektron alıcısı yöntemi kullanılmıştır. Biz phenazine methosulfate (PMS) kullanılan MPM'ler yerine onlar-si olmak benzer kimyasal makyaj ve PMS çok daha az pahalı. MPM'ler uzun bir raf ömrü ihtiyacı bir ticari kiti için önemli bir özelliği olan photochemically stabil. İstikrar sorunu olmamalı böylece Ancak, biz PMS her tahlil için taze olun. Biz de hücre dışı elektron alıcısı ve daha fazla tPMET süreci karakterize için kullanılması gereken enzimler arasındaki olası enzimatik etkileşimler (bkz. şekil 1) değerlendirmek için bir yöntem mevcut. Özellikle, AO ve SOD kullanılan enzimler tPMET kısmını askorbat taşıma veya hücre dışı süperoksit açıklaması, plazma zarı arasında taşınması elektron için iki yaygın yöntem nedeniyle belirlemek.

<table>
	<tbody>
		<tr>
			<td>C2C12 myoblasts</td>
			<td>American Type Culture Collection&#160;</td>
			<td>CRL-1772</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified eagle&#39;s medium - low glucose</td>
			<td>Sigma</td>
			<td>D6046</td>
			<td></td>
		</tr>
		<tr>
			<td>Fetal Plex animal serum complex</td>
			<td>Gemini Bio-Products&#160;</td>
			<td>100-602</td>
			<td></td>
		</tr>
		<tr>
			<td>penicillin-streptomycin</td>
			<td>Sigma</td>
			<td>516106</td>
			<td></td>
		</tr>
		<tr>
			<td>horse serum</td>
			<td>Gibco Technologies</td>
			<td>16050-130</td>
			<td></td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s phosphate buffered saline</td>
			<td>Sigma</td>
			<td>D8537</td>
			<td></td>
		</tr>
		<tr>
			<td>trypsin-EDTA</td>
			<td>Sigma</td>
			<td>T4049</td>
			<td></td>
		</tr>
		<tr>
			<td>15 cm culture dishes</td>
			<td>TPP</td>
			<td>93150</td>
			<td></td>
		</tr>
		<tr>
			<td>96 well culture plates</td>
			<td>TPP</td>
			<td>92096</td>
			<td></td>
		</tr>
		<tr>
			<td>2-(4-Iodophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazolium Sodium Salt (WST-1)</td>
			<td>Accela ChemBio&#160; Inc</td>
			<td>SY016315</td>
			<td></td>
		</tr>
		<tr>
			<td>phenazine methosulfate&#160;</td>
			<td>Sigma</td>
			<td>P9625</td>
			<td></td>
		</tr>
		<tr>
			<td>L-ascorbic acid</td>
			<td>Sigma</td>
			<td>A5960</td>
			<td></td>
		</tr>
		<tr>
			<td>ascorbate oxidase&#160;</td>
			<td>Sigma</td>
			<td>A0157</td>
			<td></td>
		</tr>
		<tr>
			<td>superoxide dismutase&#160;</td>
			<td>Sigma</td>
			<td>S5395</td>
			<td></td>
		</tr>
		<tr>
			<td>2,6-dichloroindophenol sodium salt&#160;</td>
			<td>ICN Biomedicals</td>
			<td>215011825</td>
			<td></td>
		</tr>
		<tr>
			<td>D-(+)-glucose</td>
			<td>Sigma</td>
			<td>G7528</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES sodium salt</td>
			<td>Sigma</td>
			<td>H3784</td>
			<td></td>
		</tr>
		<tr>
			<td>sodium chloride</td>
			<td>Sigma</td>
			<td>S7653</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium chloride</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP366</td>
			<td></td>
		</tr>
		<tr>
			<td>magnesium sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>M5921</td>
			<td></td>
		</tr>
		<tr>
			<td>calcium chloride dihydrate</td>
			<td>Sigma</td>
			<td>C7902</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>Fisher Scientific&#160;</td>
			<td>BP363</td>
			<td></td>
		</tr>
		<tr>
			<td>Pierce BCA Protein Assay Kit</td>
			<td>Thermo Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr>
			<td>Powerwave X-I spectrophotometer</td>
			<td>Biotek Insturments</td>
			<td>discontinued&#160;</td>
			<td></td>
		</tr>
		<tr>
			<td>Spectronic Genesys 5 Spectrophotometer</td>
			<td>Thermo Scientific</td>
			<td>336001</td>
			<td></td>
		</tr>
		<tr>
			<td>PureGrade 96-well microplate, F-bottom, clear, untreated, non-sterile</td>
			<td>MidSci</td>
			<td>781602</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) chloride tetrahydrate</td>
			<td>Sigma</td>
			<td>220299</td>
			<td></td>
		</tr>
		<tr>
			<td>Iron (II) sulfate heptahydrate</td>
			<td>Sigma</td>
			<td>215422</td>
			<td></td>
		</tr>
		<tr>
			<td>hypoxanthine</td>
			<td>Sigma</td>
			<td>H9636</td>
			<td></td>
		</tr>
		<tr>
			<td>xanthine oxidase</td>
			<td>Sigma</td>
			<td>X4500</td>
			<td></td>
		</tr>
		<tr>
			<td>Excel</td>
			<td>Microsoft</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>R Studio</td>
			<td>Rstudio</td>
			<td></td>
			<td>https://www.rstudio.com/products/rstudio/</td>
		</tr>
		<tr>
			<td>KC4</td>
			<td>Biotek Insturments</td>
			<td>discontinued&#160;</td>
			<td></td>
		</tr>
	</tbody>
</table>

measuring trans-plasma membrane electron transport by c2c12 myotubes

Trans-plazma membran elektron taşıma (tPMET) hücre dışı oksidanlar ile hücre içi indirgeyici stres hücrelerden korunması hem de hasar koruma bir rol oynar. Bu işlem için hücre dışı oksidanlar hücre içi reductants elektron taşıma iyi tanımlanmış değildir. Burada hücre dışı elektron alıcısı kullanan tPMET izlemek için C2C12 myotubes tarafından spektrofotometrik deneyleri mevcut: suda çözünen tetrazolium tuz-1 (WST-1) ve 2,6-dichlorophenolindophenol (DPIP veya DCIP). Bu elektron alıcısı azaltılması, biz gerçek zamanlı analiz bu süreçte izlemek edebiliyoruz. Enzimler askorbat oksidaz (AO) ve süperoksit dismutaz (SOD) deneyleri için gibi eklenmesiyle, biz tPMET kısmını askorbat verme ya da süperoksit üretimi nedeniyle sırasıyla belirleyebilirsiniz. WST-1 düşük arka plan ile istikrarlı sonuçlar üretmek için gösterildi iken, DPIP AO ve spektrofotometrik analiz ile gösterilmiştir SOD eklenmesi sonra yeniden oksitlenmiş olmayı başardı. Bu yöntem bir gerçek zamanlı, çok iyi, hızlı spektrofotometrik tahlil tPMET, ferricyanide (FeCN) ve ferricytochrome c azaltma gibi izlemek için kullanılan diğer yöntemler üzerinde avantajları ile gösterilmektedir.

İstatistik ANOVA ile RStudio istatistiksel yazılım25kullanarak yinelenen ölçüleri ile gerçekleştirilmiştir. Örneklerin boyutu şekil efsanelerde gösterilir.
TPMET izlemek için C2C12 myotubes hücre dışı elektron alıcısı, WST-1 ve DPIP ile birlikte kullanılmıştır. AO WST-1 kısmını belirlemek için kullanılan ve DPIP azalma nedeniyle askorbat sızma ve SOD WST-1 azaltma kısmını ...

Watch this Scientific Journal Video about Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes at JoVE.com

Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Trans-plazma membran elektron taşıma C2C12 Myotubes ile ölçme

Bu iletişim kuralı spectrophotometrically trans-plazma membran elektron taşıma ekstraselüler elektron alıcısı kullanan izlemek ve bu hücre dışı elektron alıcısı ile oluşabilir enzimatik etkileşimleri analiz için hedeftir.

Trans-plasma membrane electron transport (tPMET) plays a role in protection of cells from intracellular reductive stress as well as protection from damage ...

measuring-trans-plasma-membrane-electron-transport-by-c2c12-myotubes

Research

JoVE Journal

Biochemistry

6.8K Görüntüleme.  Saint Louis University. Bu iletişim kuralı spectrophotometrically trans-plazma membran elektron taşıma ekstraselüler elektron alıcısı kullanan izlemek ve bu hücre dışı elektron alıcısı ile oluşabilir enzimatik etkileşimleri analiz için hedeftir.

Video: Measuring Trans-Plasma Membrane Electron Transport by C2C12 Myotubes

Method to Visualize and Analyze Membrane Interacting Proteins by Transmission Electron Microscopy

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the availability of enough area to perform the function. Nanodiscs are used to provide a membrane surface of controlled size and lipid content. In the absence of bound extrinsic proteins, sodium phosphotungstate-stained nanodiscs appear as stacks of coins when viewed from the side by transmission electron microscopy (TEM). This protocol is therefore designed to intentionally promote stacking; consequently, the prevention of stacking can be interpreted as the binding of the membrane-binding protein to the nanodisc. In a further step, the TEM images of the protein-nanodisc complexes can be processed with standard single-particle methods to yield low-resolution structures as a basis for higher resolution cryoEM work. Furthermore, the nanodiscs provide samples suitable for either TEM or non-denaturing gel electrophoresis. To illustrate the method, Ca2+-induced binding of 5-lipoxygenase on nanodiscs is presented.

Many proteins perform their function when attached to membrane surfaces. The binding of extrinsic proteins on nanodisc membranes can be indirectly imaged by transmission electron microscopy. We show that the characteristic stacking (rouleau) of nanodiscs induced by the negative stain sodium phosphotungstate is prevented by the binding of extrinsic protein.

Yöntem gözünüzde canlandırın ve Transmisyon Elektron Mikroskobu Membran Etkileşen Proteinler Analiz için

Monotopic proteins exert their function when attached to a membrane surface, and such interactions depend on the specific lipid composition and on the ...

Biyokimya

Measuring Protein Binding to F-actin by Co-sedimentation

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, cross-linking and/or disassembly. This protocol describes a technique &#8211; the actin co-sedimentation, or pelleting, assay &#8211; to determine whether a protein or protein domain binds F-actin and to measure the affinity of the interaction (i.e., the dissociation equilibrium constant). In this technique, a protein of interest is first incubated with F-actin in solution. Then, differential centrifugation is used to sediment the actin filaments, and the pelleted material is analyzed by SDS-PAGE. If the protein of interest binds F-actin, it will co-sediment with the actin filaments. The products of the binding reaction (i.e., F-actin and the protein of interest) can be quantified to determine the affinity of the interaction. The actin pelleting assay is a straightforward technique for determining if a protein of interest binds F-actin and for assessing how changes to that protein, such as ligand binding, affect its interaction with F-actin.

This protocol describes a method to test the ability of a protein to co-sediment with filamentous actin (F-actin) and, if binding is observed, to measure the affinity of the interaction.

Ko-çöktürme yoluyla F-aktine Bağlı Proteinin Ölçülmesi

Filamentous actin (F-actin) organization within cells is regulated by a large number of actin-binding proteins that control actin nucleation, growth, ...

Analyzing Supercomplexes of the Mitochondrial Electron Transport Chain with Native Electrophoresis, In-gel Assays, and Electroelution

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the cell, ATP. The ETC is composed of 5 massive protein complexes, which also assemble into supercomplexes called respirasomes (C-I, C-III, and C-IV) and synthasomes (C-V) that increase the efficiency of electron transport and ATP production. Various methods have been used for over 50 years to measure ETC function, but these protocols do not provide information on the assembly of individual complexes and supercomplexes. This protocol describes the technique of native gel polyacrylamide gel electrophoresis (PAGE), a method that was modified more than 20 years ago to study ETC complex structure. Native electrophoresis permits the separation of ETC complexes into their active forms, and these complexes can then be studied using immunoblotting, in-gel assays (IGA), and purification by electroelution. By combining the results of native gel PAGE with those of other mitochondrial assays, it is possible to obtain a completer picture of ETC activity, its dynamic assembly and disassembly, and how this regulates mitochondrial structure and function. This work will also discuss limitations of these techniques. In summary, the technique of native PAGE, followed by immunoblotting, IGA, and electroelution, presented below, is a powerful way to investigate the functionality and composition of mitochondrial ETC supercomplexes.

This protocol describes the separation of functional mitochondrial electron transport chain complexes (Cx) I-V and supercomplexes thereof using native electrophoresis to reveal information about their assembly and structure. The native gel can be subjected to immunoblotting, in-gel assays, and purification by electroelution to further characterize individual complexes.

Mitokondriyal Elektron Nakil Zincirinin Süper Komplekslerinin Yerli Elektroforez, In-Jel Testleri ve Elektroelüsyon ile İncelenmesi

The mitochondrial electron transport chain (ETC) transduces the energy derived from the breakdown of various fuels into the bioenergetic currency of the ...

Semi-automated Biopanning of Bacterial Display Libraries for Peptide Affinity Reagent Discovery and Analysis of Resulting Isolates

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. Peptide affinity reagents can be used for similar applications to antibodies, including sensing and therapeutics, but are more robust and able to perform in more extreme environments. Specific enrichment of peptide capture agents to a protein target of interest is enhanced using semi-automated sorting methods which improve binding and wash steps and therefore decrease the occurrence of false positive binders. A semi-automated sorting method is described herein for use with a commercial automated magnetic-activated cell sorting device with an unconstrained bacterial display sorting library expressing random 15-mer peptides. With slight modifications, these methods are extendable to other automated devices, other sorting libraries, and other organisms. A primary goal of this work is to provide a comprehensive methodology and expound the thought process applied in analyzing and minimizing the resulting pool of candidates. These techniques include analysis of on-cell binding using fluorescence-activated cell sorting (FACS), to assess affinity and specificity during sorting and in comparing individual candidates, and the analysis of peptide sequences to identify trends and consensus sequences for understanding and potentially improving the affinity to and specificity for the target of interest.

Biopanning bacterial display libraries is a proven technique for discovery of peptide affinity reagents, a robust alternative to antibodies. The semi-automated sorting method herein has streamlined biopanning to decrease the occurrence of false positives. Here we illustrate the thought process and techniques applied in evaluating candidates and minimizing downstream analysis.

Peptid benzeşme reaktif keşif ve elde edilen yalıtır analizi için bakteriyel ekran kütüphanelerin yarı otomatik Biopanning

Biopanning bacterial display libraries is a proven technique for peptide affinity reagent discovery for recognition of both biotic and abiotic targets. ...

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM c-Cyts) has recently emerged as a novel whole-cell analytical method to characterize the bacterial electron transport from the respiratory chain to the cell exterior, referred to as the extracellular electron transport (EET). While the pathway and kinetics of the electron flow during the EET reaction have been investigated, a whole-cell electrochemical method to examine the impact of cation transport associated with EET has not yet been established. In the present study, an example of a biochemical technique to examine the deuterium kinetic isotope effect (KIE) on EET through OM c-Cyts using a model microbe, Shewanella oneidensis MR-1, is described. The KIE on the EET process can be obtained if the EET through OM c-Cyts acts as the rate-limiting step in the microbial current production. To that end, before the addition of D2O, the supernatant solution was replaced with fresh media containing a sufficient amount of the electron donor to support the rate of upstream metabolic reactions, and to remove the planktonic cells from a uniform monolayer biofilm on the working electrode. Alternative methods to confirm the rate-limiting step in microbial current production as EET through OM c-Cyts are also described. Our technique of a whole-cell electrochemical assay for investigating proton transport kinetics can be applied to other electroactive microbial strains.

Electrochemical Detection of Deuterium Kinetic Isotope Effect on Extracellular Electron Transport in Shewanella oneidensis MR-1

Here we present a protocol of whole-cell electrochemical experiments to study the contribution of proton transport to the rate of extracellular electron transport via the outer-membrane cytochromes complex in Shewanella oneidensis MR-1.

Elektrokimyasal algılama döteryum Kinetik izotop etkisi üzerinde ekstraselüler elektron taşıma Shewanella Corynebacterium Bay-1

Direct electrochemical detection of c-type cytochrome complexes embedded in the bacterial outer membrane (outer membrane c-type cytochrome complexes; OM ...

Analysis of &#946;-Amyloid-induced Abnormalities on Fibrin Clot Structure by Spectroscopy and Scanning Electron Microscopy

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and structure by spectrometry and scanning electron microscopy (SEM). A&#946;, which forms neurotoxic amyloid aggregates in Alzheimer&#39;s disease (AD), has been shown to interact with fibrinogen. This A&#946;-fibrinogen interaction makes the fibrin clot structurally abnormal and resistant to fibrinolysis. A&#946;-induced abnormalities in fibrin clotting may also contribute to cerebrovascular aspects of the AD pathology such as microinfarcts, inflammation, as well as, cerebral amyloid angiopathy (CAA). Given the potentially critical role of neurovascular deficits in AD pathology, developing compounds which can inhibit or lessen the A&#946;-fibrinogen interaction has promising therapeutic value. In vitro methods by which fibrin clot formation can be easily and systematically assessed are potentially useful tools for developing therapeutic compounds. Presented here is an optimized protocol for in vitro generation of the fibrin clot, as well as analysis of the effect of A&#946; and A&#946;-fibrinogen interaction inhibitors. The clot turbidity assay is rapid, highly reproducible and can be used to test multiple conditions simultaneously, allowing for the screening of large numbers of A&#946;-fibrinogen inhibitors. Hit compounds from this screening can be further evaluated for their ability to ameliorate A&#946;-induced structural abnormalities of the fibrin clot architecture using SEM. The effectiveness of these optimized protocols is demonstrated here using TDI-2760, a recently identified A&#946;-fibrinogen interaction inhibitor.

Presented here are two methods that can be used individually or in combination to analyze the effect of beta-amyloid on fibrin clot structure. Included is a protocol for creating an in vitro fibrin clot, followed by clot turbidity and scanning electron microscopy methods.

β-amiloid-indüklenen anormallikler Fibrin pıhtısı yapısına spektroskopisi ve elektron mikroskobu tarama Analizi

This article presents methods for generating in vitro fibrin clots and analyzing the effect of beta-amyloid (A&#946;) protein on clot formation and ...

A Fluorescence Fluctuation Spectroscopy Assay of Protein-Protein Interactions at Cell-Cell Contacts

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring cells. Of interest, only few assays are capable of specifically probing such interactions directly in living cells. Here, we present an assay to measure the binding of proteins expressed at the surfaces of neighboring cells, at cell-cell contacts. This assay consists of two steps: mixing of cells expressing the proteins of interest fused to different fluorescent proteins, followed by fluorescence fluctuation spectroscopy measurements at cell-cell contacts using a confocal laser scanning microscope. We demonstrate the feasibility of this assay in a biologically relevant context by measuring the interactions of the amyloid precursor-like protein 1 (APLP1) across cell-cell junctions. We provide detailed protocols on the data acquisition using fluorescence-based techniques (scanning fluorescence cross-correlation spectroscopy, cross-correlation number and brightness analysis) and the required instrument calibrations. Further, we discuss critical steps in the data analysis and how to identify and correct external, spurious signal variations, such as those due to photobleaching or cell movement.
In general, the presented assay is applicable to any homo- or heterotypic protein-protein interaction at cell-cell contacts, between cells of the same or different types and can be implemented on a commercial confocal laser scanning microscope. An important requirement is the stability of the system, which needs to be sufficient to probe diffusive dynamics of the proteins of interest over several minutes.

This protocol describes a fluorescence fluctuation spectroscopy-based approach to investigate interactions among proteins mediating cell-cell interactions, i.e. proteins localized in cell junctions, directly in living cells. We provide detailed guidelines on instrument calibration, data acquisition and analysis, including corrections to possible artefact sources.

Bir floresan dalgalanma spektroskopisi tahlil Protein-Protein etkileşimlerinin hücre-hücre kişiler

A variety of biological processes involves cell-cell interactions, typically mediated by proteins that interact at the interface between neighboring ...

Analysis of Protein Folding, Transport, and Degradation in Living Cells by Radioactive Pulse Chase

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and the degradation of target proteins in live cells. By using short (pulse) radiolabeling times (&lt;30 min) and tightly controlled chase times, it is possible to label only a small fraction of the total protein pool and follow its folding. When combined with nonreducing/reducing SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and immunoprecipitation with (conformation-specific) antibodies, folding processes can be examined in great detail. This system has been used to analyze the folding of proteins with a huge variation in properties such as soluble proteins, single and multi-pass transmembrane proteins, heavily N- and O-glycosylated proteins, and proteins with and without extensive disulfide bonding. Pulse-chase methods are the basis of kinetic studies into a range of additional features, including co- and posttranslational modifications, oligomerization, and polymerization, essentially allowing the analysis of a protein from birth to death. Pulse-chase studies on protein folding are complementary with other biochemical and biophysical methods for studying proteins in vitro by providing increased temporal resolution and physiological information. The methods as described within this paper are adapted easily to study the folding of almost any protein that can be expressed in mammalian or insect-cell systems.

Here we describe a protocol for a general pulse-chase method that allows the kinetic analysis of folding, transport, and degradation of proteins to be followed in live cells.

Protein katlanması, taşıma ve canlı hücreler radyoaktif darbe Chase tarafından bozulması Analizi

Radioactive pulse-chase labeling is a powerful tool for studying the conformational maturation, the transport to their functional cellular location, and ...

Interactions with and Membrane Permeabilization of Brain Mitochondria by Amyloid Fibrils

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary mechanism of amyloid aggregate-induced toxicity in neurodegenerative diseases. However, most reports describing the mechanisms of membrane disruption are based on phospholipid model systems, and studies directly targeting events occurring at the level of biological membranes are rare. Described here is a model for studying the mechanisms of amyloid toxicity at the membrane level. For mitochondrial isolation, density gradient medium is used to obtain preparations with minimal myelin contamination. After mitochondrial membrane integrity confirmation, the interaction of amyloid fibrils arising from &#945;-synuclein, bovine insulin, and hen egg white lysozyme (HEWL) with rat brain mitochondria, as an in vitro biological model, is investigated. The results demonstrate that treatment of brain mitochondria with fibrillar assemblies can cause different degrees of membrane permeabilization and ROS content enhancement. This indicates structure-dependent interactions between amyloid fibrils and mitochondrial membrane. It is suggested that biophysical properties of amyloid fibrils and their specific binding to mitochondrial membranes may provide explanations for some of these observations.

Provided here is a protocol for investigating the interactions between native form, prefibrillar, and mature amyloid fibrils of different peptides and proteins with mitochondria isolated from different tissues and various areas of the brain.

Amiloid Fibrils ile Beyin Mitokondrisi ile Etkileşimler ve Membran Permeabilizasyonu

A growing body of evidence indicates that membrane permeabilization, including internal membranes such as mitochondria, is a common feature and primary ...

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine exchangers in plants have not been identified. Here, we outline a method to characterize polyamine antiporters using membrane vesicles generated from the lysis of Escherichia coli cells heterologously expressing a plant antiporter. First, we heterologously expressed AtBAT1 in an E. coli strain deficient in polyamine and arginine exchange transporters. Vesicles were produced using a French press, purified by ultracentrifugation and utilized in a membrane filtration assay of labeled substrates to demonstrate the substrate specificity of the transporter. These assays demonstrated that AtBAT1 is a proton-mediated transporter of arginine, &#947;-aminobutyric acid (GABA), putrescine and spermidine. The mutant strain that was developed for the assay of AtBAT1 may be useful for the functional analysis of other families of plant and animal polyamine exchangers. We also hypothesize that this approach can be used to characterize many other types of antiporters, as long as these proteins can be expressed in the bacterial cell membrane. E. coli is a good system for the characterization of novel transporters, since there are multiple methods that can be employed to mutagenize native transporters.

Characterization of Membrane Transporters by Heterologous Expression in E. coli and Production of Membrane Vesicles

We describe a method for the characterization of proton-driven membrane transporters in membrane vesicle preparations produced by heterologous expression in E. coli and lysis of cells using a French press.

E. coli'de Heterolog Ekspresyon ile Membran Taşıyıcılarının Karakterizasyonu ve Membran Veziküllerinin Üretimi

Several methods have been developed to functionally characterize novel membrane transporters. Polyamines are ubiquitous in all organisms, but polyamine ...