<html> 1. Enzimatik deglycosylation <ol><li> Buharlaşma nedeniyle su kaybını en aza indirmek için PCR tüpleri kullanın. Etiket bir set borular 1 7. </li><li> Çözülme, 10X, G7 tampon, 10X glikoprotein denatüre tampon, ve% 10 NP-40 ve tüpler içeriğini karıştırmak için hafifçe dokunun. Oda sıcaklığında tutun. </li><li> Buz üzerinde enzim içeren flakon yerleştirin. Erime / donma döngüleri en aza indirmek için deneyin. </li><li> Ul dH 2 O 600 (150 mikrogram) hCGβ flakon içeriği çözülür ve buz üzerinde tutmak. </li><li> DH 2 O 10X stok seyreltilmesi 1 ml 1X G7 tampon hazırlayın </li><li> 25 ul 1X G7 tampon PNGase F 0.5 ul seyreltilir ve buz üzerinde tutmak. </li><li> 2 ul α-N-Acetylgalactosaminidase her α1-2 Fucosidase, β1-3 galaktozidaz, ve α1-3, 6 galaktosidaz birleştirerek exoglycosidase (EG karışımı) karışımı hazırlayın. </li><li> PCR tüpleri belirtildiği gibi ayarlayın: </li></ol>ak &quot;&gt; <table border="1"><tbody><tr><td> Boru / örnek # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> hCGβ (0.25mg/ml) </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> 9μl </td><td> - </td><td> - </td><td> - </td></tr><tr><td> 10X Glikoprotein Tampon denatüre </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td><td> 1μl </td></tr><tr><td> dH 2 O </td><td> - </td><td> - </td><td> - </td><td> - </td><td> 9μl </td><td> 9μl </td><td> 9μl </td></tr></tbody></table><ol start="9"><li> Tüpler kapağı, hafifçe karıştırın ve yerPCR, kapağını kapatın ve 94 az 10 dakika inkübe proteinler denatüre ° C 4 ° C tutun takip (PCR PCR tüpleri kullanılarak büyük ölçüde küçük hacimli örnekleri buharlaşmasını önler). </li><li> PCR tüpleri çıkarın ve görünür bir yoğunlaşma kaldırmak için santrifüj. </li><li> Belirtildiği gibi aşağıdaki reaktifler ekleyin: </li></ol><table border="1"><tbody><tr><td> Örnek # </td><td> 1 </td><td> 2 </td><td> 3 </td><td> 4 </td><td> 5 </td><td> 6 </td><td> 7 </td></tr><tr><td> % 10 NP-40 </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td></tr><tr><td> 10X G7 Tampon </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5μl </td><td> 2.5 ve mu; L </td><td> 2.5μl </td></tr><tr><td> PNGase F 1:50. </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td></tr><tr><td> Deglycosylation Mix </td><td> - </td><td> - </td><td> 2μl </td><td> 2μl </td><td> - </td><td> 2μl </td><td> 2μl </td></tr><tr><td> EG karışımı </td><td> - </td><td> - </td><td> - </td><td> 2μl </td><td> - </td><td> - </td><td> 2μl </td></tr><tr><td> dH 2 O </td><td> 10μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td><td> 8μl </td><td> 8μl </td><td> 6μl </td></tr><tr><td> Toplam reaksiyon vol. </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l &lt;/ Td&gt; <td> 25 μ l </td><td> 25 μ l </td><td> 25 μ l </td></tr></tbody></table><ol start="12"><li> Yeni kapaklar (bir inkübasyon döngüden sonra düzgün uymaz bu yana kullanılan at) kullanılarak PCR tüpleri kapatın. </li><li> 4 kez hafifçe dokunarak ve ardından içeriğini aşağı doğru döndürün tüpler karıştırın. </li><li> 37 ° C, 4 saat sonra 4 ° C&#39;ye kadar örnekleri serin termalcycler ve inkübe tüpleri yerleştirin </li></ol> 2. Deglycosylated örneklerin SDS-PAGE <ol><li> 4 ul 1,25 DTT ekleyerek 130 ul taze 3X azaltarak SDS yükleme tamponu hazırlayın. </li><li> Her bir örnek için hazırlanan 3X azaltarak SDS yükleme tamponu 12.5 ul ekleyin. </li><li> Yeni kapaklarla kapatın ve tüpler tüpler karıştırmak için hafifçe dokunun. </li><li> 94 bir PCR tüpleri inkübe ° C 4 ° C&#39;ye kadar serin sonra 5 dakika </li><li> Her numunenin 30 ul ve% 10-20 Tris-Glisin jel protein marker 10 ul yükleyin. Geri kalan kısmı 3.1 için örnek kaydedin. ColorPlus Prestained Protein Marker 10μl yükleyin. </li><li> 130 volt boya ön alt jel yakın olduğu kadar oda sıcaklığında jel Electrophorese. </li><li> Jel çalışması tamamlandığında, jel dökme kaldırmak ve yeterli Coomasie mavi jel kapsayacak şekilde leke küçük bir plastik kutu içine koyun. </li><li> 1 saat yavaşça sallanarak jel Leke. </li><li> Destain çözüm 50 ml, 30 dakika boyunca jel üç kez yıkayın. </li><li> Beyaz bir ışık transilluminator&#39;de veya tarayıcı kullanarak görüntüleri kaydedin. Alternatif olarak, bir çerçeve içinde selofan yaprak arasında jel kurulanmalıdır. </li></ol> 3. Pro-S Zümrüt 300 glikozile proteinler SDS-PAGE jel tespiti için <ol><li> 2.1 &#39;de jel) ile paralel olarak,% 10-20 Tris-Glisin jel örneklerinin kalanı yükleyin. Yük 10 &amp; muL ColorPlus Prestained Protein Marker. </li><li> DMF ile jel Pro-Q zümrüt reaktif çözülür ve stok Fix, yıkayın hazırlamak çalışan ve Pro-S Zümrüt 300 için çözümler Oksitleyici iken kiti ile sağlanan ürün kılavuzuna aşağıdaki leke. </li><li> Elektroforez tamamlandığında, dökme jel çıkarın ve bir plastik kutu içine koyun. </li><li> Fix çözüm 100 ml ekleyerek ve oda sıcaklığında yavaşça sallanarak o gecede terk jel sabitleyin. </li><li> Yıkama çözüm gentile ajitasyon ile oda sıcaklığında 10 ila 20 dakika için 100 ml jel yıkayın. Taze Yıkama solüsyonu ile yıkama işlemi tekrarlayın. </li><li> Çözüm Oksitleyici 25 ml 30 dakika boyunca yavaşça sallanarak jel kuluçka karbonhidratlar okside. </li><li> Adım 3.5 &#39;te açıklandığı gibi jel yıkayın. </li><li> Jel, 500 ul adım 3.2 &#39;de 25 ml çözünmüş Pro-Q Emerald 300 reaktif çözüm ekleyerek taze Pro-Q Emerald 300 leke hazırlamak yıkamatampon boyama kiti sağlanan. </li><li> Leke leke adım 3.8 &#39;de hazırlanan 25 ml ekleyerek ve 90 ila 120 dakika boyunca yavaşça sallanarak karanlıkta inkübe jel. </li><li> Adım 3.5 &#39;te açıklanan iki yıkama adımları tekrarlayın. </li><li> 300nm UV transilluminator&#39;de görüntüleri kaydedin. Pro-Q Zümrüt Yeşili ile etiketli 80 kDa işaretleyici, jel prestained merdiven gösteren bir beyaz ışık resmi UV görüntü üst üste kullanın. </li><li> Adım adım 3.11 Pro-Q zümrüt boyanan jel ile 2.10 &#39;da Coomasie lekeli jel görüntüleri karşılaştırın. </li></ol> 4. Temsilcisi sonuçları Enzimatik deglycosylation sonra protein göç değişiklikler Şekil 2&#39;de gösterilmiştir. PNGase F tedavi (N-glukanlardır kaldırılması, şerit 2) ve Deglycosylation Mix (artı endo-ve O-glukanlardır kaldırmak için exoglycosidases şeritli 3 PNGase F) (panel, şerit 1) kontrol örneği karşılaştırın. Daha fazla azalmaboyutu ek glycosidases (şeritli 4) sindirerek sonra görülür. Glukanlardır kaldırılır gibi kitlesel bir değişimin yanı sıra, bant sertleşti. 17 kDa işaretleyici (yıldız) altında çalışan bir grubun tamamen deglycosylated hCGβ polipeptid (16 kDa MW) temsil eder. Diğer bantlar, eksik deglycosylation veya hCGβ örnek mevcut çok sayıda tanımlanamayan proteinler (şeritli 1) kaynaklanıyor olabilir. Lanes 5 ila 7 (kontroller) glycosidases karşılık gelen bantları göstermektedir. Zümrüt Yeşili glikoprotein boyama Bu reaktif bir protein molekülünün tüm glukanlardır oksitler ve lekeleri, panel B&#39;de gösterilmiştir. Bu nedenle hCGβ enzimatik (şeritli 4 şeritli 1) deglycosylated olarak sinyal şiddeti azalır. 3 ve 4 şeritli artık sinyal kullanılan enzimler dayanıklı glukanıdır motifler, varlığını gösterir. Protein göç aynıdır, ancak ek glycosidases 4 şeritli olarak kullanılan bir kaç ekstra şeker artıklarını ortadan kaldırırboyanma şiddeti hafif bir azalma görülebilir. Dayanıklı şeker moieties bütün protein türlerinin mevcut değildi: yoğun deglycosylated gösteren bazı gruplar, Zümrüt Yeşil (UV görüntü yok, parantez içinde) tarafından tespit edilmedi. Ek veri hCGβ heterojen glikozile olduğu sonucuna destekler. 2 şeritli üst bant birçok glukanlardır gruplar hala mevcut olduğunu gösteren, aydınlık ise daha düşük bant şeritli 2 (ok), Zümrüt Yeşili görüntü soluk. Bu veriler, fare hücrelerinde ifade rekombinant hCGβ birden fazla glycoforms 9 içerdiğini sonuca destek . Hatta aynı protein diğerleri ise bu farklı glycoforms bazı polipeptidlerin her fikir birliği sitesi ve / veya bazı glukanlardır glukanıdır almazlar glikozilasyon doğasında heterojenite nedeniyle genişletilmiş. <img alt="Şekil 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> Şekil 1.Tipik salgılanan ya da hücre-yüzey glikoprotein glikozilasyon desenler. <img alt="Şekil 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> HCGβ enzimatik deglycosylation gösteren Şekil 2 SDS-PAGE jeller. Panel A, B Paneli glikozile proteinler görünüm için Pro-Q Emerald 300 sonuçları gösterirken, bir Coomasie mavi boyanma gösterir. Örnek sayısı: 1, hCGβ kontrolü; 2, PNGase F sindirim, 3, Deglycosylation Mix sindirim; 4, Deglycosylation Mix artı exoglycosidases sindirim, numuneler 5 ila 7 (O-Glyc, O-Glycosidase reaktif kontrolleri; TBMM, β-N Acetylglucosaminidase; Gal / sikişi, β1-3 galaktosidaz, α1-3, 6 galaktozidaz, ve α1-2 Fucosidase; GalNA, α-N-Acetylglalactosaminidase; Neu Nöraminidaz; PNGase PNGase F)

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Yazarlar, bu yazıda kullanılan reaktiflerin birçok üretir New England Biolabs tarafından istihdam edilmektedir.

Glukanıdır özel reaktifler verilerin yorumlanmasını kolaylaştırır yöntemi, enzimatik deglycosylation ve SDS-PAGE ile ilgi bir protein glikozilasyon durumu hakkında değerli bilgiler sağlayabilir Burada anlatılan. Bu protokol, protein glikozilasyon ilk çalışmaları için tasarlanmıştır ve özellikle memeli hücrelerinin salgı ve membran glikoproteinler için uygundur: Bu durumda seçilen enzimler özellikle N-glukanlardır kaldırmak ve N-glukanlardır artı ve en yaygın şekerler uzanan ve O-glukanlardır çekirdeğini oluşturan. Glycosidases hem de şeker ve protein omurga bütünlüğünü koruyarak, kimyasal deglycosylation yöntemlerle karşılaştırıldığında, hafif olmanın bir diğer avantajı var. Doluluk oranı (hangi amino asitler glikozile), glikozilasyon kapsamı, ya da ince yapısı, glukanlardır m gibi daha sofistike teknikleri belirlemek için aydınlatmak içinass spektrometresi, sıvı kromatografisi veya NMR gereklidir. Sadeliği nedeniyle, bu protokolü birkaç adımda ayarlanabilir, değiştirilemez ve / veya çeşitli deneysel ihtiyaçlarını karşılamak için kombine edilebilir. Ancak, açıkça yorumlanabilir sonuçlar elde etmek için güçlü ve sınırlamaları anlamak önemlidir. Birincisi, glycosidases özgüllüğü ve saflığı çok önemlidir: proteaz ve diğer bulaşıcı faaliyetleri ücretsiz olarak test sadece iyi karakterize enzimler kullanılmalıdır. Ne yazık ki, glycosidases için standart bir enzim birimi tanımı vardır; kullanıcı üreticinin özelliklerine göre uygun bir ikame belirlemelidir. İkincisi, tespit dikkatli bir seçim gereklidir: a) Protein boyama reaktifleri yararlıdır sadece moleküler kütlesi önemli bir vardiyada deglycosylation sonuçları. Böyle kesin bir sonuç, burada gösterildiği gibi, her zaman elde değil. Diğer durumlarda, anormal mi gördükdeglycosylation sonra biçimde entegre (tahmin molekül ağırlığı, hatta yavaş göç çok). Bu fenomen iyi anlaşılmış değildir, fakat göç herhangi bir değişiklik protein deglycosylated olduğunu kanıtlar olduğunu b) antikorları ile Şeker algılama uygulanabilirliğini sınırlayan benzersiz zorluklar sunuyor söylenebilir. Genel anti-glukanıdır antikorları üretmek için çok zor olmuştur, genellikle bunların kullanımı kısıtlayan karmaşık bir glukanıdır hedef, karşı yetiştirilir. Ayrıca, çeşitli anti-glukanıdır monoklonal antikorlar istenmeyen çapraz reaktivite 10 gösterir. C) Lektinler (içsel şeker afinite ile proteinler) ve şeker tespiti için uygundur, artı glukanıdır yapısı hakkında fikir vermek. Ancak, tüm bunların dar bir özgüllük ve birçok sadece kısmen (bilinmeyen yakınlık olabilir anlamına) karakterize edilir. Bir sonucu pozitif lektin boyama gi varlığının göstergesi değil, dayanıklı, sağlarven şeker d) Kimyasal etiketleme kitleri (şekerler periyodat oksidasyon dayalı) Tüm glikoproteinler leke için tercih edilen bir yöntemdir ve bu nedenle de deglycosylation takip etmek uygundur. Bilinmeyen bir örnek işlerken, glikoprotein kontrolleri de iyi bir uygulamadır. Fetuin hazır bir N-ve O-glikoprotein koryonik gonadotropin (her iki alt birimden) de iyi bir seçimdir. Sığır serum albumin (BSA), negatif kontrol olarak kullanılabilir. Ancak, bazı non-glikolize proteinler yüksek konsantrasyonlarda kullanıldığında, özellikle Pro-Q Emerald 300 ile biraz tepki dikkate alınmalıdır. Bu protokolde kullanılan prestained protein marker olarak non-glikolize moleküler ağırlık standartları, keskin bantları görüntüleme avantajı var. Sadece şans eseri bu proteinlerin (80 kDa), Pro-Q Emerald 300 reaktif biridir. Bu nedenle, kullanıcı gibi Invitroge Şeker Kamışı gibi bir glikoprotein standart merdiven yerine çalıştırmak için isteyebilirsinizn. Son olarak, 11 özgüllük kontrolü için β-N-Acetylglucosaminidase boyunca nucleocytosolic glikoproteinler (tek bir O-GlcNAc ile değiştirilmiş olan) basit jel algılama, monoklonal antikor kullanımı ile mümkündür, bu tekniğin açıklaması ötesinde Bu makale kapsamında, ama glycosidases hücrelerinde mevcut diğer birçok glikoproteinler ve glycoconjugates çalışma için yararlı araçlar olarak belirtilmelidir. 12:: NCBI Kitaplık (20.301.239 [NBK1908 Kitaplık ID) ücretsiz çevrimiçi; glikozilasyon bilinen tüm formları kapsamlı bir tedavi &quot;Glikobiyoloji Essentials&quot; ikinci baskısında bulunabilir.

<table border="1"><tbody><tr><td> Reaktifi Adı </td><td> Şirket </td><td> Katalog numarası </td><td> Yorumlar </td></tr><tr><td> İnsan koryonik gonadotropin β </td><td> Sigma Aldrich </td><td> C6572 </td><td></td></tr><tr><td> PCR Tüpleri </td><td> VWR </td><td> 20170-004 </td><td></td></tr><tr><td> PCR PCR </td><td> Applied Biosystems </td><td> 4359659 </td><td></td></tr><tr><td> PNGase F </td><td> New England Biolabs </td><td> Den P0704&#39;e </td><td> Tamponlar ile sağlanır </td></tr><tr><td> Protein Deglycosylation Mix </td><td> New England Biolabs </td><td> P6039 </td><td> Tamponlar ile sağlanır </td></tr><tr><td> α-N-Acetylglalactosaminidase </td><td> New England Biolabs </td><td> P0734 </td><td> Tamponu ile sağlanır </td></tr><tr><td> α1-2 Fucosidase </td><td> New England Biolabs </td><td> P0724 </td><td> Tamponu ile sağlanır </td></tr><tr><td> α1-3, 6 galaktosidaz </td><td> New England Biolabs </td><td> P0731 </td><td> Tamponu ile sağlanır </td></tr><tr><td> β1-3 galaktosidaz </td><td> New England Biolabs </td><td> P0726 </td><td> Tamponu ile sağlanır </td></tr><tr><td> G7 10X Buffer (500 mM Sodyum Fosfat) </td><td> New England Biolabs </td><td> --- </td><td> PNGaseF veya Degl Mix ile birlikte verilir </td></tr><tr><td> 10X Glikoprotein denatüre Tampon (% 5 SDS, 400 mM DTT) </td><td> New England Biolabs </td><td> --- </td><td> PNGaseF veya Degl Mix ile birlikte verilir </td></tr><tr><td> % 10 NP-40 </td><td> New England Biolabs </td><td> --- </td><td> PNGaseF veya Degl Mix ile birlikte verilir </td></tr><tr><td> 3X SDS yükleme tamponu (187.5 mM Tris-HCl, pH 6.8,% 6 SDS,% 30 gliserol,% 0.03 yabanibademophenol mavi) </td><td> New England Biolabs </td><td> B7703 </td><td> 1,25 DTT ile birlikte verilir </td></tr><tr><td> ColorPlus Protein Marker Prestained </td><td> New England Biolabs </td><td> P7709 </td><td></td></tr><tr><td> % 10-20 Tris-Glisin Multigel </td><td> Cosmo Bio Co </td><td> DCB-414.893 </td><td></td></tr><tr><td> Kaset Elektroforez Ünitesi </td><td> Cosmo Bio Co </td><td> DCB-303.111 </td><td></td></tr><tr><td> Elektroforez Güç Kaynağı EPS 301 </td><td> GE Healthcare </td><td> 18-1130-01 </td><td></td></tr><tr><td> Pro-Q Emerald 300 Glikoprotein Stain Kit </td><td> Invitrogen </td><td> P-21857 </td><td></td></tr><tr><td> Brillant Blue R </td><td> Sigma Aldrich </td><td> B0149 </td><td></td></tr><tr><td> AlphaImager HP Sistemi </td><td> Hücre Biosciences </td><td> 92-13823-00 </td><td></td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glikozilasyon, kovalent bağlarla bağlanmış şekerlerin yanı sıra, proteinlerin hücre adezyon, moleküler kaçakçılığı, gümrükleme, ve sinyal iletimi 1-4 gibi süreçleri önemli ölçüde etkileyebilir post-translasyonel önemli bir değişiklik . Veya serin veya treonin artıkları (O bağlantılı) (Şekil 1); ökaryotlar, salgı yolağı en yaygın glikozilasyon değişiklikler konsensüs asparajin artıkları (N-bağlantılı) ilave. N-glukanıdır sentezinin başlatılması son derece farklı türler, doku veya protein son ürünleri arasında çok büyük farklılıklar ise, ökaryotlarda korunur . Bazı değiştirilmemiş glukanlardır (&quot;yüksek mannoz K-glukanlardır&quot;) ya da daha fazla Golgi (&quot;karmaşık K-glukanlardır&quot;) işlenir kalır. Hayvan hücrelerinde ortak bir N-Acetylgalactosamine (GalNAc) kalıntı ile başlar ama daha aşağı organizmalardan 1 farklı O-glukanlardır, büyük çeşitlilik bulundu. ent &quot;&gt; kendi başına bir alan proteinlerin glikozilasyon detaylı analiz ve doğru çalışabilmesi için geniş kaynaklar ve uzmanlık gerektirir. kaldır şekerler (glycosidases) glikozilasyon durumu hakkında genel bir fikir mümkün kılan mevcut enzimlerin Ancak çeşitli Burada standart bir laboratuar ortamında bir protein analizi için bir model glikoprotein glycosidases kullanımı göstermektedir: iki K-glukanlardır ve dört O glukanlardır 5 taşıyan rekombinant insan koryonik gonadotropin beta (hCGβ), tekniği, sadece basit gerektirir enstrümantasyon ve tipik ve sarf malzemeleri, birden fazla glikoprotein örnekleri analiz için kolayca adapte edilebilir. Bazı enzimler bir glikoprotein çalışma paralel olarak kullanılabilir. PNGase F N-bağlı glukanlardır 6,7 hemen hemen her türlü kaldırmak mümkün. O-glukanlardır için bir enzim yoktur tutunmaya th sağlam bir oligosakkarite protein omurgası. Bunun yerine, O-glukanlardır kısa bir çekirdek, sonra O-Glycosidase kolayca kaldırılır exoglycosidases tarafından kesilmiş bulunmaktadır. Protein Deglycosylation Mix PNGase Nöraminidaz F, O Glycosidase, (sialidase), β1-4 galaktosidaz ve β-N-Acetylglucosaminidase içerir. Aynı anda K-glukanlardır ve bazı O-glukanlardır 8 kaldırmak için kullanılır. Son olarak, Deglycosylation Mix, aksi takdirde mevcut olabilir dayanıklı Monosakkaritler kaldırmanıza yardımcı diğer exoglycosidases bir karışımı (6 galaktosidaz α1-3, α1-2 Fucosidase, α-N-Acetylgalactosaminidase, β1-3 galaktosidaz) ile tamamlanıyor belirli Ç glukanlardır. SDS-PAGE/Coomasie mavi glycosidase tedavi öncesi ve sonrası protein göç farklılıkları görselleştirmek için kullanılır. Glukanlardır succe Buna ek olarak, şeker özel bir boyama yöntemi, ProQ zümrüt-300, azalmış sinyal gösterirssively kaldırıldı. Bu protokol, enzimatik deglycosylation gerektiği kadar büyük miktarlarda protein karşılamak için ölçeklendirilebilir, ancak küçük miktarlarda glikoprotein (0,5-2 mg) analizi için tasarlanmıştır.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

SDS-PAGE ile takip glikoproteinler şekerler kaldırmak için belirli glycosidases kullanarak protein örnekleri üzerinde glukanıdır değişiklikler tespit etmek için değerli bir yöntemdir ve ilk Glikobiyoloji çalışmaları için iyi bir seçimdir. Deglycosylation aşağıdaki değişiklikler, jel hareketlilik vardiya olarak veya glukanıdır duyarlı reaktifler boyama ile tespit edilebilir.

Spesifik endo-ve exoglycosidases kullanarak protein glikozilasyon tanımlanması ve karakterizasyonu

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

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36.4K Görüntüleme.  New England Biolabs. SDS-PAGE ile takip glikoproteinler şekerler kaldırmak için belirli glycosidases kullanarak protein örnekleri üzerinde glukanıdır değişiklikler tespit etmek için değerli bir yöntemdir ve ilk Glikobiyoloji çalışmaları için iyi bir seçimdir. Deglycosylation aşağıdaki değişiklikler, jel hareketlilik vardiya olarak veya glukanıdır duyarlı reaktifler boyama ile tespit edilebilir.

Video: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach. 

Here we describe a new quantitative proteomics technique for identifying protein complexes in Saccharomyces cerevisiae. In this study, we have used the SILAC method together with affinity purification followed by tandem mass spectrometry to identify with high specificity the binding partners of an ER protein, Scs2p.

S. cerevisiae, kantitatif proteomik protein kompleksleri tanımlanması

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as ...

Biyoloji

Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based fractionation. The system is comprised of single-use, 8-sample capacity cartridges and a benchtop GELFREE Fractionation Instrument. During separation, a constant voltage is applied between the anode and cathode reservoirs, and each protein mixture is electrophoretically driven from a loading chamber into a specially designed gel column gel. Proteins are concentrated into a tight band in a stacking gel, and separated based on their respective electrophoretic mobilities in a resolving gel. As proteins elute from the column, they are trapped and concentrated in liquid phase in the collection chamber, free of the gel. The instrument is then paused at specific time intervals, and fractions are collected using a pipette. This process is repeated until all desired fractions have been collected. If fewer than 8 samples are run on a cartridge, any unused chambers can be used in subsequent separations.
This novel technology facilitates the quick and simple separation of up to 8 complex protein mixtures simultaneously, and offers several advantages when compared to previously available fractionation methods. This system is capable of fractionating up to 1mg of total protein per channel, for a total of 8mg per cartridge. Intact proteins over a broad mass range are separated on the basis of molecular weight, retaining important physiochemical properties of the analyte. The liquid phase entrapment provides for high recovery while eliminating the need for band or spot cutting, making the fractionation process highly reproducible1.

The accompanying video describes the use of the GELFREE 8100 Fractionation System, which partitions complex protein samples on the basis of molecular weight and recovers the fractions in liquid phase. The video describes how the technology works, how it is used, and provides resultant data, with polyacrylamide gel electrophoresis analysis of fractionated bovine liver homogenate.

Sıvı Faz Kurtarma Moleküler Ağırlık Tabanlı Fraksiyonu GELFREE 8100 Fraksiyonu Sisteminin Kullanılması

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Identification of Protein Interacting Partners Using Tandem Affinity Purification

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein partners. There are many approaches that allow the identification of interacting partners, including the yeast two hybrid system, as well as pull down assays using recombinant proteins and immunoprecipitation of endogenous proteins followed by mass spectrometry identification1. Recent studies have highlighted the utility of double-affinity tag mediated purification, coupled with two specific elution steps in the identification of interacting proteins. This approach, termed Tandem Affinity Purification (TAP), was initially used in yeast2,3 but more recently has been adapted to use in mammalian cells4-8.
As proof-of-concept we have established a tandem affinity purification (TAP) method using the well-characterized eukaryotic translation initiation factor eIF4E9,10.The cellular translation factor eIF4E is a critical component of the cellular eIF4F complex involved in cap-dependent translation initiation10. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence. The TAP tag used in the current study is composed of two Protein G units and a streptavidin binding peptide separated by a Tobacco Etch Virus (TEV) protease cleavage sequence8. To forgo the need for the generation of clonal cell lines, we developed a rapid system that relies on the expression of the TAP-tagged bait protein from an episomally maintained plasmid based on pMEP4 (Invitrogen). Expression of tagged murine eIF4E from this plasmid was controlled using the cadmium chloride inducible metallothionein promoter.
Lysis of the expressing cells and subsequent affinity purification via binding to rabbit IgG agarose, TEV protease cleavage, binding to streptavidin linked agarose and subsequent biotin elution identified numerous proteins apparently specific to the eIF4E pull-down (when compared to control cell lines expressing the TAP tag alone). The identities of the proteins were obtained by excision of the bands from 1D SDS-PAGE and subsequent tandem mass spectrometry. The identified components included the known eIF4E binding proteins eIF4G and 4EBP-1. In addition, other components of the eIF4F complex, of which eIF4E is a component were identified, namely eIF4A and Poly-A binding protein. The ability to identify not only known direct binding partners as well as secondary interacting proteins, further highlights the utility of this approach in the characterization of proteins of unknown function.

Tandem affinity purification is a robust approach for the identification of protein binding partners. As proof of concept, this methodology was applied to the well-characterized translation initiation factor eIF4E to co-precipitate the host cell factors involved in translation initiation. This method is easily adapted to any cellular or viral protein.

Tandem Affinity Arıtma kullanarak Protein etkileşim Ortaklar belirlenmesi

A critical and often limiting step in understanding the function of host and viral proteins is the identification of interacting cellular or viral protein ...

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

Kompleks Örnekleri spesifik protein Glikozilasyon arasında Multiplexed Yüksek verim Profilleme için Kimyasal-bloke Antikor Mikroarray

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

Method for the Isolation and Identification of mRNAs, microRNAs and Protein Components of Ribonucleoprotein Complexes from Cell Extracts using RIP-Chip

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and readily available form of data collection. In many research and disease models however, steady state levels of target gene mRNA does not always directly correlate with steady state protein levels. Post-transcriptional gene regulation is a likely explanation of the divergence between the two. Driven by the binding of RNA Binding Proteins (RBP), post-transcriptional regulation affects mRNA localization, stability and translation by forming a Ribonucleoprotein (RNP) complex with target mRNAs. Identifying these unknown de novo mRNA targets from cellular extracts in the RNP complex is pivotal to understanding mechanisms and functions of the RBP and their resulting effect on protein output. This protocol outlines a method termed RNP immunoprecipitation-microarray (RIP-Chip), which allows for the identification of specific mRNAs associated in the ribonucleoprotein complex, under changing experimental conditions, along with options to further optimize an experiment for the individual researcher. With this important experimental tool, researchers can explore the intricate mechanisms associated with post-transcriptional gene regulation as well as other ribonucleoprotein interactions.

A step by step protocol to isolating and identifying RNA associated complexes through RIP-Chip.

RIP-Chip kullanarak Hücre Ekstraktlarının gelen mRNA mikroRNA&#39;lar ve ribonükleoprotein Kompleksi Protein Bileşenlerinin İzolasyonu ve Tanımlama için Yöntemi

As a result of the development of high-throughput sequencing and efficient microarray analysis, global gene expression analysis has become an easy and ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

Protein Kompleksi belirlenmesi<em&gt; Escherichia coli</em&gt; Tandem Kütle Spektrometresi ile Kombine Sıralı Peptid Affinity Arıtma kullanarak

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Generation, Purification, and Characterization of Cell-invasive DISC1 Protein Species

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases Alzheimer's disease (A&beta;, tau), Parkinson's Disease (&alpha;-synuclein), Huntington's disease (polyglutamine, huntingtin), and others. Protein aggregation is thought to occur due to disturbed proteostasis, i.e. the imbalance between the arising and degradation of misfolded proteins. Of note, the same proteins are found aggregated in sporadic forms of these diseases that are mutant in rare variants of familial forms.
Schizophrenia is a chronic progressive brain condition that in many cases goes along with a permanent and irreversible cognitive deficit. In a candidate gene approach, we investigated whether Disrupted-in-schizophrenia 1 (DISC1), a gene cloned in a Scottish family with linkage to chronic mental disease1, 2, could be found as insoluble aggregates in the brain of sporadic cases of schizophrenia3. Using the SMRI CC, we identified in approximately 20 % of cases with CMD but not normal controls or patients with neurodegenerative diseases sarkosyl-insoluble DISC1 immunoreactivity after biochemical fractionation. Subsequent studies in vitro revealed that the aggregation propensity of DISC1 was influenced by disease-associated polymorphism S704C4, and that DISC1 aggresomes generated in vitro were cell-invasive5, similar to what had been shown for A&beta;6, tau7-9, &alpha;-synuclein10, polyglutamine11, or SOD1 aggregates12. These findings prompted us to propose that at least a subset of cases with CMD, those with aggregated DISC1 might be protein conformational disorders.
 Here we describe how we generate DISC1 aggresomes in mammalian cells, purify them on a sucrose gradient and use them for cell-invasiveness studies. Similarly, we describe how we generate an exclusively multimeric C-terminal DISC1 fragment, label and purify it for cell invasiveness studies. Using the recombinant multimers of DISC1 we achieve similar cell invasiveness as for a similarly labeled synthetic &alpha;-synuclein fragment. We also show that this fragment is taken up in vivo when stereotactically injected into the brain of recipient animals.

The generation, purification and cell invasion of intracellular, cytoplasmic full length DISC1 protein aggresomes from cell cultures and of a labeled, multimeric recombinant DISC1 protein fragment in E. coli are described. Cell invasiveness is shown for recipient cells in cell culture and for neurons in vivo after stereotactical brain inoculation.

Hücre-invaziv DISC1 Protein Türlerinin Üretimi, Saflaştırılması, ve Karakterizasyonu

Protein aggregation is seen as a general hallmark of chronic, degenerative brain conditions like, for example, in the neurodegenerative diseases ...

Pyrosequencing for Microbial Identification and Characterization

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains and detect genetic mutations that confer resistance to anti-microbial agents. The advantages of pyrosequencing for microbiology applications include rapid and reliable high-throughput screening and accurate identification of microbes and microbial genome mutations. Pyrosequencing involves sequencing of DNA by synthesizing the complementary strand a single base at a time, while determining the specific nucleotide being incorporated during the synthesis reaction. The reaction occurs on immobilized single stranded template DNA where the four deoxyribonucleotides (dNTP) are added sequentially and the unincorporated dNTPs are enzymatically degraded before addition of the next dNTP to the synthesis reaction. Detection of the specific base incorporated into the template is monitored by generation of chemiluminescent signals. The order of dNTPs that produce the chemiluminescent signals determines the DNA sequence of the template. The real-time sequencing capability of pyrosequencing technology enables rapid microbial identification in a single assay. In addition, the pyrosequencing instrument, can analyze the full genetic diversity of anti-microbial drug resistance, including typing of SNPs, point mutations, insertions, and deletions, as well as quantification of multiple gene copies that may occur in some anti-microbial resistance patterns.

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate bacterial strains, and detect genetic mutations that confer resistance to anti-microbial agents. In this video, the procedure for microbial amplicon generation, amplicon pyrosequencing, and DNA sequence analysis will be demonstrated.

Mikrobiyal Tanımlama ve Karakterizasyonu için Pyrosequencing

Pyrosequencing is a versatile technique that facilitates microbial genome sequencing that can be used to identify bacterial species, discriminate ...

Isolation of Specific Genomic Regions and Identification of Associated Molecules by enChIP

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. To enable the non-biased identification of molecules interacting with a specific genomic region of interest, we recently developed the engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) technique. Here, we describe how to use enChIP to isolate specific genomic regions and identify the associated proteins and RNAs. First, a genomic region of interest is tagged with a transcription activator-like (TAL) protein or a clustered regularly interspaced short palindromic repeats (CRISPR) complex consisting of a catalytically inactive form of Cas9 and a guide RNA. Subsequently, the chromatin is crosslinked and fragmented by sonication. The tagged locus is then immunoprecipitated and the crosslinking is reversed. Finally, the proteins or RNAs that are associated with the isolated chromatin are subjected to mass spectrometric or RNA sequencing analyses, respectively. This approach allows the successful identification of proteins and RNAs associated with a genomic region of interest.

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the functions of these regions. This protocol describes procedures to perform engineered DNA-binding molecule-mediated chromatin imunoprecipitation (enChIP) for identification of proteins and RNAs associated with a specific genomic region.

Belirli Genomik Bölgeler ve enChIP tarafından İlişkili Moleküllerin Tanımlanması İzolasyonu

The identification of molecules associated with specific genomic regions of interest is required to understand the mechanisms of regulation of the ...

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

Elevated lipogenesis is a common characteristic of cancer and metabolic diseases. Sterol regulatory element-binding proteins (SREBPs), a family of membrane-bound transcription factors controlling the expression of genes important for the synthesis of cholesterol, fatty acids and phospholipids, are frequently upregulated in these diseases. In the process of SREBP nuclear translocation, SREBP-cleavage activating protein (SCAP) plays a central role in the trafficking of SREBP from the endoplasmic reticulum (ER) to the Golgi and in subsequent proteolysis activation. Recently, we uncovered that glucose-mediated N-glycosylation of SCAP is a prerequisite condition for the exit of SCAP/SREBP from the ER and movement to the Golgi. N-glycosylation stabilizes SCAP and directs SCAP/SREBP trafficking. Here, we describe a protocol for the isolation of membrane fractions in human cells and for the preparation of the samples for the detection of SCAP N-glycosylation and total protein by using western blot. We further provide a method to monitor SCAP trafficking by using confocal microscopy. This protocol is appropriate for the investigation of SCAP N-glycosylation and trafficking in mammalian cells.

Analysis of SCAP N-glycosylation and Trafficking in Human Cells

We describe a modified method for membrane fraction isolation from human cells and sample preparation for the detection of SCAP N-glycosylation and total protein by using western blot. We further introduce a GFP-labeling method to monitor SCAP trafficking using confocal microscopy. This protocol can be used in regular biology laboratories.