Bu çalışma, ABD Ulusal Bilim Vakfı Christoph Benning bir hibe ile desteklenmektedir.

1. Lipid Ekstraksiyon <ol><li> 30 mg agar üzerinde yetiştirilen bitkiler 4 hafta eski Arabidopsis yaprakları orta ya da toprak katılaşmış ve 1,5 mL polipropilen reaksiyon tüpleri içine transfer hasat tarafından lipid ekstraksiyon başlayın. Taze yaprakları flaş sıvı nitrojen içinde dondurulmuş ve -80 ° C&#39;de saklanabilir. </li><li> Metanol oluşan solvent 300 mcL çıkarma, kloroform ve formik asit (20:10:01, v / v / v) her numune ekleyin. (Bir boya Shaker ya da benzeri kullanarak) 5 dakika çalkalanır. </li><li> 0.2 M fosforik asit (H 3 PO 4), 1 M potasyum klorür (KCl) ve vorteks kısaca 150 mcL ekleyin. </li><li> 1 dakika boyunca oda sıcaklığında 13.000 xg&#39;de santrifüjleyin. Alt kloroform fazında çözünmüş Lipidler TLC plakaları damlatılan olacaktır. </li></ol> 2. İnce Tabaka Kromatografisi (TLC) 15 <ol><li> Batığın 0.15 M amonyum sülfat ((NH 4) 2 SO 4) solüsyonu içine 30 saniye için şerit yükleme 20cmX20cm ile silika jel kaplı TLC plağı, TLC plakaları hazırlamak için, 30 saniye boyunca daldırarak sonra, en az 2 gün boyunca tabak kuru kapalı bir kapta. Etkinleştirme sırasında diğer glycerolipids ayrılması için gerekli phosphatidylglycerol protonates sülfürik asit, amonyum süblimasyon arkasında bırakır. </li><li> Deney günü etkinleştirmek 120 fırında pişirme tarafından plakaları TLC ° C, 2,5 saat süreyle. </li><li> Oda sıcaklığına kadar aktive edilmiş plakalar Soğuduktan sonra, plaka üzerinde kromatogram kökeni (plaka kenarından 1,5 cm) düz bir çizgi çizmek için bir kalem kullanın. </li><li> Davlumbaz, yavaş yavaş bir akışı altında N 2 200 mcL sarı plastik ipuçları ile 20 mcL pipet kullanarak alt kloroform faz 3 X 20 mcL lipid özü teslim. Bu amaçla, bir Tygon Boru N 2 tank, regülatör bağlıdır. Spot çapı 1 cm&#39;den daha küçük tutunuz. Her plaka 10 örnek (sonraki GLC analiz planlanan olduğunda) kadar tutabilir. </li><li> Lipid davlumbaz tamamen kuru noktalar gibi, aseton, toluen, su (30 ml: 7.5 ml 91 ml) oluşan gelişmekte olan solvent hazırlar. Ortam bağıl nem yüksek ise, ayrılık etkilenebilir. Bu durumda istenen ayırım elde (7.0 ml: 30 ml 91 ml) su vermek azaltılmalıdır. </li><li> 80 ml çözücü gelişmekte olan ve aşağı bakacak şekilde örnek sonuna tankına plaka odası (H: W = 27.0: 26.5 7.0 cm / cm / cm L) gelişmekte olan bir yapışmalı, TLC içine dökün. Tank kelepçe kullanarak Seal. Solvent plaka yükselmek ve lipidler ayrılacaktır. Geliştirme zamanını, oda sıcaklığında yaklaşık 50 dakika mesafededir. </li><li> Çözücü ön plaka üstten 1 cm ulaştığında, dikkatlice plaka tanktan çıkarın ve yaklaşık 10 dakika için davlumbaz tamamen kuru. </li><li> TLC ayrılmış Lipidler kantitatif analiz ya da sülfürik asit veya α-naftol geri dönüşümsüz lekeli iyot ile ya da geri dönüşümlü kısaca lekeli olabilir. <ol><li> Sülfürik asit yanmaları:% 50 sülfürik asit, davlumbaz ve fırında 120 ° C&#39;de 15 dakika (Şekil 2A) spreyli cam bir şişede su spreyi plaka. </li><li> glikolipitler için α-naftol boyama:% 2.4 (w / v)% 10 α-naftol (v / v), sülfürik asit,% 80 (v / v) etanol ve fırında 120 ° C plaka uygulaması için 3-5 glikolipid bantları kadar dakika (Şekil 2B), pembe veya mor lekeli. Aşırıtedavi reaktif sülfürik asit varlığı nedeniyle tüm lipidler yanmaları için yol açacaktır. </li><li> İyot boyaması: davlumbaz plaka, iyot kristalleri ile kapalı bir TLC (lipidler görünür olana kadar iyot buharı ile atmosferin doygunluk önde gelen altındaki bir tepsi) tankı içine yerleştirin. Iyot iyot kovalent çoklu doymamış yağ asitleri (Şekil 2C) değiştirebilir gibi çok uzun plaka maruz bırakmayın. Alternatif olarak, örnek şeritli serpiştirilmiş sadece standart şerit N 2 ayrı standart şerit üzerinde üflenir iyot kristalleri ile Pasteur pipeti takılı bir cam yünü kullanılarak boyandı olmalıdır, lipidlerin herhangi bir oksidasyon önlemek için. </li></ol></li></ol> 3. Yağ Asil metilester (FAME) Reaksiyon 16 <ol><li> Bir jilet ile TLC plağı silika çevreleyen lipit noktalar tespit çıkarın. Lipit içeren silika kazıyın ve Teflon (PTFE) kaplı vidalı kapaklı cam tüp içine bir huni kullanarak silis tozu aktarımı. </li><li> Cam pipet ile her bir örnek susuz metanol 1 ml 1 N hidroklorik asit (HCl) ekleyin. </li><li> 200 mcL sarı plastik ucu ile 200 mcL pipet kullanarak iç standart olarak her numune için 200 mcL pipet kullanarak 100 mcL 50 mg mL -1 pentadecanoic asit (15:00) ekleyin. Bir tüp kontrol olarak sadece pentadecenoic metanol HCl asit ile tutun. Cam tüpler Teflon kaplı kapakları sıkıca kapatın. </li><li> Inkübe gl25 dakika boyunca 80 ° C su banyosu göt tüpleri. Solvent buharlaşma olmaz böylece Tüpler mühürlü olması gerekir. </li><li> Tüpler soğutulduktan sonra, şiddetle 1 ml hekzan ve vorteks 1 ml% 0.9 sodyum klorür ekleyin. 3 dakika için 1000xg numune santrifüjleyin. </li><li> Davlumbaz, Pasteur pipeti ile örnek hekzan / üst tabakayı kaldırmak ve yeni bir 13x100 mm cam tüp içine yerleştirin. </li><li> N 2 yavaş bir akışı tamamen kuruması beklenmeden altında hekzan buharlaşır. </li><li> 60 mcL hekzan çıkan yağ açil methylesters s eritin. Örnekleri otosampler şişeleri ve kapağı sıkıca içine aktarın. Numuneler, kısa vadeli ve -20 ° C ° C bir kaç gün için 4 saklanabilir. </li></ol> 4. Gaz-Sıvı Kromatografi (GLC) 10 <ol><li> GLC başlamadan önce, helyum, hidrojen ve hava tüpleri dolu olduğundan emin olun. </li><li> Yeterli hekzan, solvent rezervuar eklendi olmalı ve atık kabını boş olmalıdır. Yağ açil methylesters ayrılması için, makineyi bir DB-23 sütun ekleyebilirsiniz. </li><li> Otosampler yerleştirmek gözlerdir. Sistemi bilgisayar GLC ChemStation yazılımını başlatın. </li><li> Giriş sıcaklığı 250 ° C helyum akış hızı ve 48.6 mL dak -1 21.93 psi basınç. Ayarlayın Split oranı 30.0: 1. </li><li> Fırın sıcaklığı 140 ° C &#39;de 2 dakika süreyle başlangıçta ortaya konan ve 160 ° C - 25 ° C min -1 hızında kaldırdı . Ardından 160 artış sıcaklığını ayarlamak ° C ila 250 ° C hızında 8 ° C min -1 ve 250 tutun ° C - 140 azalma takip 4 dk ° C hızında 38 ° C min - 1. Bir vadede yaklaşık 21 dakika sürer. </li><li> Alev iyonizasyon dedektör sıcaklığı 270 ° C ve hidrojen debisi 30.0 mL dak -1, hava debisi 400 mL dak -1 ve helyum debisi 30.0 mL dak -1. </li><li> Şişeleri ve çalışma sıra tabloda örnek isimleri numarasını girin. 10 mcL enjektör, flakon başına 2 mcL örnek enjekte etmek için ayarlayın. </li><li> Cihaz hazır olduğunda, çalışma dizisi başlatmak. </li></ol> 5. Temsilcisi Sonuçlar: 4 haftalık Arabidopsis fide TLC ayrılmış lipitlerin geri dönüşü olmayan boyama örnekleri Şekil 2&#39;de gösterilmiştir. Sülfürik asit lekeli lipidler (Şekil 2A) kömürleşmiş ve kahverengi lekeler olarak görünür. α-naftol diğer polar lipid (Şekil 2B) sarı leke α-naftol pembe-mor bir renk taşıyan boyanmış vb Glikolipitler SQDG DGDG gibi MGDG glikolipitler leke tercih edilir. Iyot boyama geri dönüşümlüdür ve lipidler, iyot buharlaşır (Şekil 2C) gibi kısa bir zaman içinde kaybolur sarımsı bir renk verir. Lekesiz lipidler Lipidlerin yıkmak azaltmak için tercih edilir rağmen Kısaca iyot lekeli lipidler GLC analiz tabi tutulabilir. Başarılı bir şekilde yapılırsa, farklı Yağ açil metilester temsil eden ayırt edici sinyaller (Şekil 3) GLC sonra gözlenen olacaktır. Yağ açil metilester kısa karbon zinciri ve daha az çift bağ ile DB-23 sütun kullanarak kısa tutma süresi var. Yağ açil metilester profil değişmiş lipid kompozisyonu ile mutantlar tanımlamak için hassas bir araçtır. Şekil 4&#39;te, vahşi tip 18 ile karşılaştırıldığında tgd4-1 mutant MGDG18: 3 yağ asidi molar oranda azalmıştır. Yağ açil metilester bir lipid sınıf için tüm lipid sınıflarının mol mol bölerek, her lipid molar oranı hesaplanır. Örneğin, MGDG molar oranını hesaplamak için: (MGDG) mol% = Σ [Fames (MGDG)] / Σ [Fames (toplam)] x100% Yabani tip ve mutant her lipid sınıfı molar oranları mukayese edilebilir. Örneğin, tgd4-1 mutant MGDG ve PG göreceli miktarları artmış ama DGDG ve PE (Şekil 5) 18 miktarda azalma var. <img src="/files/ftp_upload/2518/2518fig1.jpg" alt="Şekil 1" /> Şekil 1 Arabidopsis fide kullanarak polar lipit analizi akış şeması. Toplam lipidler, 4 haftalık Arabidopsis fide çıkarılan ve TLC ayrılır . Ayrılmış lipidler GLC analizi ile takip transesterifikasyon TLC plağı kazınmış olabilir. <img src="/files/ftp_upload/2518/2518fig2.jpg" alt="Şekil 2" /> Şekil 2 TLC plakaları üzerinde lipitlerin ayrılması. 35 mg lipid özleri (taze ağırlık) vahşi tip fide TLC ayrılmış ve sülfürik asit (A), α-naftol (B) veya iyot buharı (C) ile boyanan. Üç tekrarlar, her boyama yöntemi gösterilmiştir. DGDG, digalactosyldiacylglycerol; MGDG, monogalactosyldiacylglycerol; PC, fosfatidilkolin, PE, phosphatidylethanolamine; PG, phosphatidylglycerol; PI, fosfatidilinositol; SQDG, sulfoquinovosyldiacylglycerol. <img src="/files/ftp_upload/2518/2518fig3.jpg" alt="Şekil 3" /> Şekil 3 Yağ Asitleri Methylesters GLC analizi (Fames) vahşi tip MGDG türetilmiştir. Fames 30 m kılcal sütun üzerinde ayrılır ve alev iyonizasyon tarafından tespit edilir. Pentadecanoic asit (15:00) bir iç standart olarak kullanılır. <img src="/files/ftp_upload/2518/2518fig4.jpg" alt="Şekil 4" /> Şekil 4 vahşi tip Süt2 (beyaz sütunlar) ve mutant tgd4-1 (siyah sütunlar) MGDG Yağ asidi profili. Yağ asitleri çift bağların sayısına göre takip karbonlar sayısı olarak sunulmaktadır. Üç tekrarlar ortalama ve standart sapmaları gösterilmiştir. <img src="/files/ftp_upload/2518/2518fig5.jpg" alt="Şekil 5" /> Şekil 5 vahşi tip Süt2 (beyaz sütunlar) Polar lipidler kompozisyon ve tgd4-1 mutant (siyah sütunlar). Üç tekrarlar ortalama ve standart sapmalar hata bar gösterilmiştir.

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TLC GLC ile birleştiğinde tesislerinde polar lipid kantitatif analiz için sağlam ve hızlı bir araç sağlar. Lipid bileşimi küçük değişiklikler tespit edilebilir, bu nedenle bu yöntem, polar lipid metabolik yollar 1,20 engelli mutantlar büyük ölçekli tarama için kullanılır olmuştur . Bu yöntem ayrıca, substrat olarak polar lipidler kullanan enzimler izleme faaliyetleri için yaygın olarak kullanılır . 2,6,7 Yaprakları yanı sıra, bu kök ve tohumları veya kloroplast ve mitokondri gibi hücre içi fraksiyonları gibi diğer bitki dokularının lipid kompozisyonu da aynı şekilde tespit edilebilir. Burada kullanılan çözücü sistemi (aseton, toluen, su), bitkilerde glikolipitler ve fosfolipidlerin ayrılması için optimize edilmiştir. Ancak, tgd1, 2,3,4 mutantlar ve izole kloroplastların tetragalactosyldiacylglycerol PC ile çalışırken TGDG PE ile birlikte çalışır. Bu durumda, kloroform, metanol, asetik asit ve su (85: 20: 10: 4, v / v / v / v) ile çözücü sistem 13 kullanılır . Bazen iki farklı çözücü sistemlerini kullanarak iki boyutlu TLC, daha fazla ayrı glikolipitler ve fosfolipitler 19 yapılır . Buna ek olarak, bitki dokularında doğrudan TLC 5 başlangıç ​​ayrımı olmadan toplam yağ asidi profili belirlemek için GLC takip FAME reaksiyon tabi olabilir. Gösterdi TLC GLC sisteminin yanı sıra, lipid profili kullanılan başka bir yöntem, doğrudan elektrosprey iyonizasyon tandem kütle spektrometresi 17 dayanmaktadır . Bu yöntemde ilk kromatografik ayırma özü lipitlerin atlanır. Ancak, bu yöntem, laboratuvar veya mutant taraması için rutin analizler için daha az kullanışlı hale getirir pahalı ekipman ve deneyimli personel gerektirir.

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		<th>Material Name</th>
		<th>Type</th>
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		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>&alpha;-naphthol</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>N1000</td>
<td>&nbsp;</td>
<tr>
<td>Methanolic HCL 3N</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>33050-U</td>
<td>Dilute to 1N by methanol</td>
</tr>
<tr>
<td>Si250-PA TLC plates</td>
<td>&nbsp;</td>
<td>J.T.Baker</td>
<td>7003-04</td>
<td>With pre-absorbent </td>
</tr>
<tr>
<td>TLC chamber</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>Z266000</td>
<td>&nbsp;</td>
<tr>
<td>Screw cap tubes</td>
<td>&nbsp;</td>
<td>VWR</td>
<td>53283-800</td>
<td>&nbsp;</td>
<tr>
<td>Scew caps</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>13-425</td>
<td>&nbsp;</td>
<tr>
<td>PTFE disk</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>200 608</td>
<td>&nbsp;</td>
<tr>
<td>GLC system</td>
<td>&nbsp;</td>
<td>Hewlett Packard</td>
<td>HP6890</td>
<td>&nbsp;</td>
<tr>
<td>DB-23 column</td>
<td>&nbsp;</td>
<td>J&amp;W Scientific</td>
<td>122-2332</td>
<td>&nbsp;</td>
<tr>
<td>GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>500 132</td>
<td>&nbsp;</td>
<tr>
<td>Caps of GLC vials</td>
<td>&nbsp;</td>
<td>Sun Sri</td>
<td>201 828</td>
<td>&nbsp;</td>
<tr>
<td>Chemstation software</td>
<td>&nbsp;</td>
<td>Agilent</td>
<td>G2070AA</td>
<td>&nbsp;</td>		</tbody>
		</table>
		</div>

arabidopsis thaliana polar glycerolipid profiling by thin layer chromatography (tlc) coupled with gas-liquid chromatography (glc)

Biyolojik membranlar ayrı hücreler çevreden. Fosfatidilkolin veya phosphatidylethanolamine, hücreleri ve çevreleri arasındaki kimyasal değişimi için hem sınırları ve arayüzleri olarak hareket form bilayeri membranlar gibi çok hücreli bitki ve hayvanlar, glycerolipids, tek bir hücre. Hayvanların aksine, bitki hücreleri fotosentez için özel bir organel kloroplast var. (MGDG) monogalactosyldiacylglycerol digalactosyldiacylglycerol (DGDG), sulfoquinovosyldiacylglycerol (SQDG) 4: kloroplastın karmaşık membran sistemi yani benzersiz glycerolipids, fosfor eksikliği glikolipitler içerir. Bu lipitlerin rolleri sadece yapısal dışındadır. Bu glikolipitler ve diğer glycerolipids fotosistem I ve II fotosentez 8,11 glycerolipids katılımı gösteren kristal yapıları bulundu. Fosfat açlık sırasında, DGDG fosfolipitler 9,12 kaybını telafi etmek extraplastidic membranlar aktarılır. Bu lipitlerin biyosentezi ve fonksiyon bilgimizin Arabidopsis thaliana 14 ile biyokimyasal ve genetik çalışmalar bir arada elde edilmiştir. Bu çalışmalar sırasında, polar lipid analizi için basit bir prosedür lipid mutantlar tarama ve analiz için gerekli ve detaylı olarak özetlenen olacaktır. Bir yaprak lipid özü ilk ince tabaka kromatografisi (TLC) ile ayrılmış ve glycerolipids iyot buharı ile geri dönüşümlü lekeli. Bireysel lipidler TLC plağı koparılmış ve alev iyonizasyon algılama (FID GLC) (Şekil 1) ile birleştiğinde gaz-sıvı kromatografisi ile analiz edilir yağ açil methylesters (Fames) dönüştürülür. Bu yöntem, mutant tarama için güvenilir bir araç olduğu kanıtlanmıştır. Örneğin tgd1, 2,3,4 endoplazmik retikulum-plastid lipid kaçakçılığı mutantlar anormal bir galactoglycerolipid birikimine dayalı bulunmuştur: trigalactosyldiacylglycerol (TGDG) ve göreli 18:03 miktarda bir azalma (karbonlar: double bağ) 3,13,18,20 zarı lipidler yağ açil grupları. Bu yöntem ayrıca substrat 6 olarak lipidler kullanarak proteinlerin enzimatik faaliyetleri belirlemek için uygulanabilir.

Watch this Scientific Journal Video about Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC at JoVE.com

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

Polar lipid özler bireysel glycerolipids Kompozisyon ve yağ asidi kompozisyonu basit ve sağlam bir lipid profil deneyde belirlenir. Bu amaçla, glycerolipids ince tabaka kromatografisi ile izole edilir ve onların açil grupları transmethylation tabi. Yağ açil methylesters gaz-sıvı kromatografisi ile sayısallaştırılmış.

İnce Tabaka Kromatografisi (TLC) tarafından Arabidopsis thaliana Polar Glycerolipid Profil Gaz-Sıvı Kromatografi (GLC) ile birleştiğinde

Biological membranes separate cells from the environment. From a single cell to multicellular plants and animals, glycerolipids, such as ...

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Research

JoVE Journal

Biology

Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography (TLC) Coupled with Gas-Liquid Chromatography (GLC)

36.6K Görüntüleme.  Michigan State University. Polar lipid özler bireysel glycerolipids Kompozisyon ve yağ asidi kompozisyonu basit ve sağlam bir lipid profil deneyde belirlenir. Bu amaçla, glycerolipids ince tabaka kromatografisi ile izole edilir ve onların açil grupları transmethylation tabi. Yağ açil methylesters gaz-sıvı kromatografisi ile sayısallaştırılmış.

Video: Arabidopsis thaliana Polar Glycerolipid Profiling by Thin Layer Chromatography TLC Coupled with Gas-Liquid Chromatography GLC

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Numune Hazırlama: Ultra İnce Tabaka Yöntemi

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biyoloji

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts from different plant tissues was first reported more than 40 years ago 1 and has since been adapted to study a variety of cellular processes, such as subcellular localization of proteins, isolation of intact organelles and targeted gene-inactivation by double stranded RNA interference (RNAi) 2-5. Most of the protoplast isolation protocols use leaf tissues of mature Arabidopsis (e.g. 35-day-old plants) 2-4. We modified existing protocols by employing 14-day-old Arabidopsis seedlings. In this procedure, one gram of 14-day-old seedlings yielded 5 106-107 protoplasts that remain intact at least 96 hours. The yield of protoplasts from seedlings is comparable with preparations from leaves of mature Arabidopsis, but instead of 35-36 days, isolation of protoplasts is completed in 15 days. This allows decreasing the time and growth chamber space that are required for isolating protoplasts when mature plants are used, and expedites the downstream studies that require intact protoplasts.

Isolation of Protoplasts from Tissues of 14-day-old Seedlings of Arabidopsis thaliana

This video shows a procedure for isolating intact protoplasts from tissues of 14-day-old seedlings of Arabidopsis. Given that the isolated protoplasts remain intact for at least 96h and are isolated from seedlings instead of one-month-old mature plants, this procedure expedites assays requiring intact protoplasts.

14 gün eski Fideler Dokular protoplast izolasyonu Arabidopsis thaliana</em

Protoplasts are plant cells that have had their cell walls enzymatically removed. Isolation of protoplasts  from different plant tissues was first ...

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikor Profil Lusiferaz Immunoprecipitation Sistemleri (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

Dayanıksız Multi-protein Kompleksleri izolasyonu In vivo Kontrollü Hücresel Çapraz Bağlama ve immuno-manyetik Afinite Kromatografisi

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called photoreceptors to sense and adapt to light. The red/far-red light-absorbing phytochrome photoreceptors have been studied extensively. Phytochromes exist as a family of proteins with distinct and overlapping functions in all higher plant systems in which they have been studied1. Phytochrome-mediated light responses, which range from seed germination through flowering and senescence, are often localized to specific plant tissues or organs2. Despite the discovery and elucidation of individual and redundant phytochrome functions through mutational analyses, conclusive reports on distinct sites of photoperception and the molecular mechanisms of localized pools of phytochromes that mediate spatial-specific phytochrome responses are limited. We designed experiments based on the hypotheses that specific sites of phytochrome photoperception regulate tissue- and organ-specific aspects of photomorphogenesis, and that localized phytochrome pools engage distinct subsets of downstream target genes in cell-to-cell signaling. We developed a biochemical approach to selectively reduce functional phytochromes in an organ- or tissue-specific manner within transgenic plants. Our studies are based on a bipartite enhancer-trap approach that results in transactivation of the expression of a gene under control of the Upstream Activation Sequence (UAS) element by the transcriptional activator GAL43. The biliverdin reductase (BVR) gene under the control of the UAS is silently maintained in the absence of GAL4 transactivation in the UAS-BVR parent4. Genetic crosses between a UAS-BVR transgenic line and a GAL4-GFP enhancer trap line result in specific expression of the BVR gene in cells marked by GFP expression4. BVR accumulation in Arabidopsis plants results in phytochrome chromophore deficiency in planta5-7. Thus, transgenic plants that we have produced exhibit GAL4-dependent activation of the BVR gene, resulting in the biochemical inactivation of phytochrome, as well as GAL4-dependent GFP expression. Photobiological and molecular genetic analyses of BVR transgenic lines are yielding insight into tissue- and organ-specific phytochrome-mediated responses that have been associated with corresponding sites of photoperception4, 7, 8. Fluorescence Activated Cell Sorting (FACS) of GFP-positive, enhancer-trap-induced BVR-expressing plant protoplasts coupled with cell-type-specific gene expression profiling through microarray analysis is being used to identify putative downstream target genes involved in mediating spatial-specific phytochrome responses. This research is expanding our understanding of sites of light perception, the mechanisms through which various tissues or organs cooperate in light-regulated plant growth and development, and advancing the molecular dissection of complex phytochrome-mediated cell-to-cell signaling cascades.

Investigating Tissue- and Organ-specific Phytochrome Responses using FACS-assisted Cell-type Specific Expression Profiling in Arabidopsis thaliana

The molecular basis of spatial-specific phytochrome responses is being investigated using transgenic plants that exhibit tissue- and organ-specific phytochrome deficiencies. The isolation of specific cells exhibiting induced phytochrome chromophore depletion by Fluorescence-Activated Cell Sorting followed by microarray analyses is being utilized to identify genes involved in spatial-specific phytochrome responses.

Hücre tipi Özgül gösterilmeden FACS yardımlı kullanarak Doku ve Organ spesifik Fitokrom Yanıtları incelenmesi Arabidopsis thaliana</em

Light mediates an array of developmental and adaptive processes throughout the life cycle of a plant. Plants utilize light-absorbing molecules called ...

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&beta;-glucuronidase Reporter Gene Expression

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis thaliana, the floral-dip transformation method1-2 has replaced all previous methods because of its simplicity, efficiency, and low cost. Specifically, shoots of young flowering Arabidopsis plants are dipped in a solution of Agrobacterium tumefaciens carrying specific plasmid constructs. After dipping, the plants are returned to normal growth and yield seeds, a small percentage of which are transformed with the foreign gene and can be selected for on medium containing antibiotics. This floral-dip method significantly facilitated Arabidopsis research and contributed greatly to our understanding of plant gene function. In this study, we use the floral-dip method to transform a reporter gene, &beta;-glucuronidase (GUS), under the control of TSO2 promoter. TSO2, coding for the Ribonucleotide Reductase (RNR) small subunit3, is a cell cycle regulated gene essential for dNDP biosynthesis in the S-phase of the cell cycle. Examination of GUS expression in transgenic Arabidopsis seedlings shows that TSO2 is expressed in actively dividing tissues. The reported experimental method and materials can be easily adapted not only for research but also for education at high school and college levels.

Floral-dip Transformation of Arabidopsis thaliana to Examine pTSO2::&#946;-glucuronidase Reporter Gene Expression

This article illustrates the floral-dip method of Agrobacterium tumefaciens -mediated transformation of Arabidopsis thaliana. By introducing a cell-cycle regulated promoter-reporter, pTSO2::&beta;-glucuronidase (GUS), into Arabidopsis, we illustrates how one detects GUS reporter expression in transgenic seedlings.

Floral-daldırma Dönüşüm Arabidopsis thaliana Inceleyin PTSO2: β-glucuronidase Reporter Gen İfadesi

The ability to introduce foreign genes into an organism is the foundation for modern biology and biotechnology. In the model flowering plant Arabidopsis ...

Automated Hydrophobic Interaction Chromatography Column Selection for Use in Protein Purification

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction chromatography (HIC) commonly requires experimental determination (referred to as screening or "scouting") in order to select the most suitable chromatographic medium for purifying a given protein 1. The method presented here describes an automated approach to scouting for an optimal HIC media to be used in protein purification.
HIC separates proteins and other biomolecules from a crude lysate based on differences in hydrophobicity. Similar to affinity chromatography (AC) and ion exchange chromatography (IEX), HIC is capable of concentrating the protein of interest as it progresses through the chromatographic process. Proteins best suited for purification by HIC include those with hydrophobic surface regions and able to withstand exposure to salt concentrations in excess of 2 M ammonium sulfate ((NH4)2SO4). HIC is often chosen as a purification method for proteins lacking an affinity tag, and thus unsuitable for AC, and when IEX fails to provide adequate purification. Hydrophobic moieties on the protein surface temporarily bind to a nonpolar ligand coupled to an inert, immobile matrix. The interaction between protein and ligand are highly dependent on the salt concentration of the buffer flowing through the chromatography column, with high ionic concentrations strengthening the protein-ligand interaction and making the protein immobile (i.e. bound inside the column) 2. As salt concentrations decrease, the protein-ligand interaction dissipates, the protein again becomes mobile and elutes from the column. Several HIC media are commercially available in pre-packed columns, each containing one of several hydrophobic ligands (e.g. S-butyl, butyl, octyl, and phenyl) cross-linked at varying densities to agarose beads of a specific diameter 3. Automated column scouting allows for an efficient approach for determining which HIC media should be employed for future, more exhaustive optimization experiments and protein purification runs 4.
The specific protein being purified here is recombinant green fluorescent protein (GFP); however, the approach may be adapted for purifying other proteins with one or more hydrophobic surface regions. GFP serves as a useful model protein, due to its stability, unique light absorbance peak at 397 nm, and fluorescence when exposed to UV light 5. Bacterial lysate containing wild type GFP was prepared in a high-salt buffer, loaded into a Bio-Rad DuoFlow medium pressure liquid chromatography system, and adsorbed to HiTrap HIC columns containing different HIC media. The protein was eluted from the columns and analyzed by in-line and post-run detection methods. Buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection increased the functionality of the system and reproducibility of the experimental approach.

An automated method for identifying suitable hydrophobic interaction chromatography (HIC) media to be used in the process of protein purification is presented. The method utilizes a medium-pressure liquid chromatography system including automated buffer blending, dynamic sample loop injection, sequential column selection, multi-wavelength analysis, and split fraction eluate collection.

Protein Saflaştırma Kullanım için Otomatik Hidrofobik Etkileşim Kromatografisi Sütun Seçimi

In contrast to other chromatographic methods for purifying proteins (e.g. gel filtration, affinity, and ion exchange), hydrophobic interaction ...

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a consequence, the isolation of plant embryos at early stages (1 cell to globular stage) is technically challenging due to their relative inaccessibility. Efficient manual dissection at early stages is strongly impaired by the small size of young Arabidopsis seeds and the adhesiveness of the embryo to the surrounding tissues. Here, we describe a method that allows the efficient isolation of young Arabidopsis embryos, yielding up to 40 embryos in 1 hr to 4 hr, depending on the downstream application. Embryos are released into isolation buffer by slightly crushing 250-750 seeds with a plastic pestle in an Eppendorf tube. A glass microcapillary attached to either a standard laboratory pipette (via a rubber tube) or a hydraulically controlled microinjector is used to collect embryos from droplets placed on a multi-well slide on an inverted light microscope. The technical skills required are simple and easily transferable, and the basic setup does not require costly equipment. Collected embryos are suitable for a variety of downstream applications such as RT-PCR, RNA sequencing, DNA methylation analyses, fluorescence in situ hybridization (FISH), immunostaining, and reporter gene assays.

Efficient and Rapid Isolation of Early-stage Embryos from Arabidopsis thaliana Seeds

We report an efficient and simple method to isolate embryos at early stages of development from Arabidopsis thaliana seeds. Up to 40 embryos can be isolated in 1 hr to 4 hr, depending on the downstream application. The procedure is suitable for transcriptome, DNA methylation, reporter gene expression, immunostaining and fluorescence in situ hybridization analyses.

Erken evre Embriyolar verimli ve hızlı İzolasyon<em&gt; Arabidopsis thaliana</em&gt; Tohumlar

In flowering plants, the embryo develops within a nourishing tissue - the endosperm - surrounded by the maternal seed integuments (or seed coat). As a ...

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling processes. Extracting sterol-enriched membrane microdomains from plant cells for proteomic analysis is a difficult task mainly due to multiple preparation steps and sources for contaminations from other cellular compartments. The plasma membrane constitutes only about 5-20% of all the membranes in a plant cell, and therefore isolation of highly purified plasma membrane fraction is challenging. A frequently used method involves aqueous two-phase partitioning in polyethylene glycol and dextran, which yields plasma membrane vesicles with a purity of 95% 1. Sterol-rich membrane microdomains within the plasma membrane are insoluble upon treatment with cold nonionic detergents at alkaline pH. This detergent-resistant membrane fraction can be separated from the bulk plasma membrane by ultracentrifugation in a sucrose gradient 2. Subsequently, proteins can be extracted from the low density band of the sucrose gradient by methanol/chloroform precipitation. Extracted protein will then be trypsin digested, desalted and finally analyzed by LC-MS/MS. Our extraction protocol for sterol-rich microdomains is optimized for the preparation of clean detergent-resistant membrane fractions from Arabidopsis thaliana cell cultures.
We use full metabolic labeling of Arabidopsis thaliana suspension cell cultures with K15NO3 as the only nitrogen source for quantitative comparative proteomic studies following biological treatment of interest 3. By mixing equal ratios of labeled and unlabeled cell cultures for joint protein extraction the influence of preparation steps on final quantitative result is kept at a minimum. Also loss of material during extraction will affect both control and treatment samples in the same way, and therefore the ratio of light and heave peptide will remain constant. In the proposed method either labeled or unlabeled cell culture undergoes a biological treatment, while the other serves as control 4.

Metabolic Labeling and Membrane Fractionation for Comparative Proteomic Analysis of Arabidopsis thaliana Suspension Cell Cultures

Here we describe a robust method for the fractionation of plant plasma membranes into detergent resistant and detergent soluble membranes based on a mixture of unlabeled and in vivo fully 15N labeled Arabidopsis thaliana cell cultures. The procedure is applied for comparative proteomic studies to understand signaling processes.

Mukayeseli Proteomik Analizi Metabolik Etiketleme ve Membran Fraksiyonasyon<em&gt; Arabidopsis thaliana</em&gt; Süspansiyon Hücre Kültürleri

Plasma membrane microdomains are features based on the physical properties of the lipid and sterol environment and have particular roles in signaling ...

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively in the study of secondary cell wall formation. Secondary cell wall deposition occurs after the primary cell wall is laid down, a process carried out exclusively by specialized cells such as those forming vessel and fiber tissues. Most secondary cell walls are composed of cellulose (40&#8211;50%), hemicellulose (25&#8211;30%), and lignin (20&#8211;30%). Several mutations affecting secondary cell wall biosynthesis have been isolated, and the corresponding mutants may or may not exhibit obvious biochemical composition changes or visual phenotypes since these mutations could be masked by compensatory responses. Staining procedures have historically been used to show differences on a cellular basis. These methods are exclusively visual means of analysis; nevertheless their role in rapid and critical analysis is of great importance. Congo red and calcofluor white are stains used to detect polysaccharides, whereas M&#228;ule and phloroglucinol are commonly used to determine differences in lignin, and toluidine blue O is used to differentially stain polysaccharides and lignin. The seemingly simple techniques of sectioning, staining, and imaging can be a challenge for beginners. Starting with sample preparation using the A. thaliana model, this study details the protocols of a variety of staining methodologies that can be easily implemented for observation of cell and tissue organization in secondary cell walls of plants.

Histochemical Staining of Arabidopsis thaliana Secondary Cell Wall Elements

Plant cell wall composition varies between tissue types and can include lignin, cellulose, hemicelluloses, and pectin. Various staining techniques have been developed to visualize differences at the cell-type level. This paper is a compilation of commonly used cell wall staining techniques.

Histokimyasal boyama<em&gt; Arabidopsis thaliana</em&gt; Ortaöğretim Hücre Duvar Elemanları

Arabidopsis thaliana is a model organism commonly used to understand and manipulate various cellular processes in plants, and it has been used extensively ...