1. Örnek Protein Hazırlık <ol><li> 500 ul mikrofuge&#39;de tüp, aşağıdaki bir araya getirir: </li></ol><table border="1"><tbody><tr><td> Reaktif </td><td> Eklemek için Cilt: </td></tr><tr><td> * Protein örneği (1 mg) </td><td> 112 mcL </td></tr><tr><td> Kartuş kiti sağlanan örnek Tampon (5X) </td><td> 30 mcL </td></tr><tr><td> DTT 1M </td><td> 8μL </td></tr><tr><td> 18 Ω H 2 O </td><td> x 150μL örnek hacminin toplam mcL ** </td></tr></tbody></table><ol start="2"><li> Örnekleri 95 ° C&#39;de 5 dakika denatüre. Oda sıcaklığında soğutun. </li></ol> * Ayrılmış protein toplam miktarın örnek karmaşıklığına bağlı olacaktır. , Doku Homojenat gibi daha karmaşık örnekleri, için, kanal başına 150-200 mg tavsiye edilir . Örnek hacmi 112 mcL az **, 150 mcL nihai hacmi getirmek için 18 Ω H 2 O ekleyin. 2. GELFREE Kartuş Yükleniyor <ol><li> GELFREE 8100 Kartuşu folyo poşetten çıkarın. </li><li> Plaka macunu çıkarın ve atın. </li><li> Bir pipet kullanarak kartuşu bölmesine depolama tampon çıkarın. Kartuş 8 odaları kullanılacaksa, kartuşu saklama bölmeleri tampon drenaj ters olabilir. </li><li> Her kartuş odaları için aşağıdaki gibi GELFREE Koşu Tampon ekleyerek kartuşu yükleme için hazırlayın: </li></ol><table border="1"><tbody><tr><td> GELFREE Kartuş Odası </td><td> Ses eklemek GELFREE Koşu Tampon </td></tr><tr><td> Anot tampon haznesi </td><td> 8 mL </td></tr><tr><td> Katot tampon rezervuar </td><td> 6 ml </td></tr><tr><td> Koleksiyon odaları </td><td> Her biri 100 mcL </td></tr></tbody></table><ol start="5"><li> Örnek bir yükleme bölmesine herhangi bir tampon çıkarıp atın. </li><li> 8 kanallı pipet kullanarak yükleme odalarına 150 mcL örnekleri yükleyin. </li><li> GELFREE 8100 Fraksiyonu Station içine yüklenen kartuş yerleştirin. Elektrot dizileri indirin ve kapağını kapatın. </li></ol> 3. Kartuş Koşu <ol><li> Cihazın dokunmatik ekran kullanarak, önceden programlanmış yöntemleri listesini görmek için yöntem düğmesine basın. Önceden programlanmış yöntemleri Her proteinlerin molekül ağırlıkları belirli bir aralık içinde çözmek için tasarlanmış belirlenen gerilimler ve zaman bazlı duraklar oluşur. GELFREE 8100 fraksiyon toplanması için yöntem belirtilen aralıklarla otomatik olarak ara. </li><li> Uygun bir yöntem tespit edildikten sonra, geri alma tuşuna basın ve ekrandaki tuş takımını kullanarak istediğiniz önceden programlanmış bir yöntem numarasını girin . OK tuşuna basın, daha sonra uygula düğmesine basın. Uygulanan yöntem, ana ekranın alt kısmında görünecektir. </li><li> Vadede kullanılacak kanallar (numune sayısı) seçmek için, ana ekranda kanal düğmesine basın. Sekiz kanal seçmek için, tümünü seç tuşuna basın. Daha sonra, ana ekrana dönmek için yapılan basın. </li><li> Emniyet kapağı kapalı olduğundan emin olun ve gösterge ışığı ekranın altındaki yeşil olduğunu. </li><li> Protein ayırma başlatmak için, start tuşuna basın. Kesirler Not toplamak için zamanı geldiğinde Fraksiyonu İstasyonu duraklama otomatik olarak sistem çalışırken, ekranın sol tarafındaki kutuya gösterilen çevreler her kanalın durumunu tasvir. Yeşil bir daire kanalı aktif olduğunu gösterir, sarı bir daire, enstrüman duraklatılmış olduğunu gösterir ve kırmızı bir daire kanalı başarısız olduğunu gösterir . Uygulanan gerilim ve akımların kanal durumu çevrelerin sağ görüntülenir. Durumunu çevrelerin açık sarı ve bir metin uyarı alet s ekranda göründüğünde Kesirler sadece çıkarılmalıdır. </li><li> Kesirler kaldırmak için, cihazın kapağını açın ve toplama odalarının her birinden 150 mcL çekmek için 8 kanallı pipet kullanın. </li><li> Kesirler çıkarıldıktan sonra, kanal başına 100 ul ekleyerek ve iki kez yukarı ve aşağı pipetleme GELFREE Koşu Tampon toplama haznesi iki kez yıkayın. </li><li> Yıkadıktan sonra, toplama odalarına geri Tampon Koşu GELFREE 100uL ekleyin, kapağını kapatın, ve devam düğmesine basın . </li><li> GELFREE 8100 sonraki zaman aralığı kadar sürecek. Deneyde kalan toplam süre ekranın sağ tarafında gösterilir. Kalan süre arasında geçiş yapmak için, bir süre geçtikten ve duraklama zamanı, zaman gösteren kutusunun üzerindeki düğmeye basın . </li><li> Bir kere bütün fraksiyonlarının ortak olmuşturllected, hemen izoelektrik odaklama veya jel elektroforezi gibi daha fazla downstream hazırlanması, veya likit kromatografi kütle spektrometresi veya immünoafinite kullanarak analizi için kullanılan olabilir. </li></ol> 4. Temsilcisi Sonuçlar GELFREE Fraksiyonu Sistemi kullanılarak, her birine 12 kesirler sığır karaciğer Homojenat 200 mg bölündü. Her fraksiyonu 6 mcL bir standart 1D SDS PAGE jel ve gümüş boyanan çalıştırıldı. Şekil 1&#39;de gösterildiği gibi, protein örneği 3.5kDa 150kDa için moleküler ağırlık aralığı kapsayan, 12 farklı fraksiyonları fraksiyone oldu. 1 ve 14 şeritli bir moleküler ağırlık standardı (ColorBurst Sigma) gösterilmiştir. Burada gösterilen jel protein fraksiyonasyon GELFREE 8100 sistemini kullanarak elde edilen yüksek iyileşme oranı vurgulamaktadır. Burada gösterilen 200 mg kurtarıldı protein jel% 4 oranında temsil eder. <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="Şekil 1" border="0" /> Şekil 1. Sığır karaciğer Proteinler GELFREE Fraksiyonu sistemi kullanılarak ayrılır Homojenat. Yüklenen her 150 mcL fraksiyonu 6 mcL ve 1D SDS PAGE jel ve gümüş boyanan çalıştırıldı. Belirtildiği gibi bir molekül ağırlığı (MW) standardı da yüklendi. Lütfen Şekil 1&#39;de bir büyük halini görmek için <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">buraya tıklayınız</a> .

<ol>
	<li>Tran, J. C., Doucette, A. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiplexed+Size+Separation+of+Intact+Proteins+in+Solution+Phase+for+Mass+Spectrometry.">Multiplexed Size Separation of Intact Proteins in Solution Phase for Mass Spectrometry.</a> Anal Chem.. , (2009).</li></ol>

Bu makalede kullanılan reaktifler ve aletleri üreten Protein Discovery bu makalenin yazarları tarafından istihdam edilmektedir.

GELFREE 8100 Fraksiyonu Sistemi ayrık molekül ağırlığı tabanlı kesirler karmaşık protein örneklerinin bölümleme için hızlı, basit ve tekrarlanabilir bir yöntem sağlar. Fraksiyonasyon Bu yeni bir yöntem sayesinde 90 dakika içinde geniş bir kütle aralığı üzerinde proteinleri izole etmek için, bant veya spot kesme ihtiyacını ortadan kaldırır. Örnek nispeten büyük miktarlarda yük ve sıvı faz fraksiyonları kurtarmak için tipik olarak algılanması daha zor olan düşük bolluk proteinler, izolasyonu ve analiz yeteneği yararlıdır. Ortaya çıkan kesirler sonra geniş bir uygulama dizisi, LC-MS, MALDI-TOF ve immunoblotting gibi kullanılabilir.

using the gelfree 8100 fractionation system for molecular weight-based fractionation with liquid phase recovery

GELFREE 8100 Fraksiyonu Sistemi, molekül ağırlığı fraksiyonasyon sırasında protein kurtarma en üst düzeye çıkarmak için tasarlanmış olan yeni bir protein fraksiyonasyon sistemi. Bu sistem, tek kullanımlık, 8-örnek kapasiteli kartuşlar ve benchtop GELFREE Fraksiyonu Enstrüman oluşur. Ayrılması sırasında, anot ve katot rezervuarları arasında sabit bir gerilim uygulanır ve her protein karışımı elektroforetik bir yükleme bölmesine özel tasarlanmış jel kolon jel sürülür. Proteinler, bir yığın jel sıkı bir bant içine konsantre ve çözme jel kendi elektroforetik hareketlilik dayalı ayrılır. Proteinler sütun Zehir gibi, onlar toplama odasında jel sıkışıp kalmasına ve sıvı faz yoğunlaşmıştır. Cihaz sonra belirli zaman aralıklarında duraklatılmış ve kesirler bir pipet kullanılarak toplanan. İstenen tüm fraksiyonları toplanıncaya kadar bu işlem tekrarlanır. Az 8 örnek bir kartuş üzerinde çalışan varsa, kullanılmayan odaları sonraki ayrımlarında kullanılabilir. Bu yeni bir teknoloji, aynı anda 8 kompleks protein karışımları kadar hızlı ve basit bir ayrımı kolaylaştırır ve daha önce mevcut fraksiyon yöntemleri kıyasla birçok avantaj sunar. Bu sistem kartuş başına 8mg toplam 1mg kadar kanal başına total protein fraksiyonasyon yeteneğine sahiptir. Geniş bir kütle aralığı içinde bozulmamış proteinler istinat analitin önemli fizikokimyasal özellikleri, molekül ağırlığı esasına göre ayrılır. 1 fraksiyonasyon süreci son derece tekrarlanabilir bant veya spot kesme ihtiyacını ortadan kaldırarak, sıvı faz tuzak yüksek iyileşme sağlar.

Watch this Scientific Journal Video about Using the GELFREE 8100 Fractionation System for Molecular Weight-Based Fractionation with Liquid Phase Recovery at JoVE.com

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

İlişikteki video GELFREE 8100 Fraksiyonu Sistemi kullanımı, kompleks protein örneklerinin moleküler ağırlık bazında bölümleri açıklar ve sıvı faz fraksiyonları kurtarır. Video teknoloji, nasıl kullanılır, inşaat işleri ve fraksiyone sığır karaciğer Homojenat poliakrilamid jel elektroforezi analizleri ile ortaya çıkan veri sağlar.

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 ...

using-gelfree-8100-fractionation-system-for-molecular-weight-based

Research

JoVE Journal

Biology

16.3K Görüntüleme.  Protein Discovery, Inc.. İlişikteki video GELFREE 8100 Fraksiyonu Sistemi kullanımı, kompleks protein örneklerinin moleküler ağırlık bazında bölümleri açıklar ve sıvı faz fraksiyonları kurtarır. Video teknoloji, nasıl kullanılır, inşaat işleri ve fraksiyone sığır karaciğer Homojenat poliakrilamid jel elektroforezi analizleri ile ortaya çıkan veri sağlar.

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

Preparation and Fractionation of Xenopus laevis Egg Extracts

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical analysis. Egg lysis and differential centrifugation are used to prepare the crude extract which in turn in used to prepare fractionated extracts and light membrane preparations.

Xenopus laevis Yumurta hazırlanması ve Fraksiyonu ayıklar.

Crude and fractionated Xenopus egg extracts can be used to provide ingredients for reconstituting cellular processes for morphological and biochemical ...

Biyoloji

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, many proteins are present as a freely diffusing population in free exchange with a sub-population that is tightly associated with a particular subcellular domain or structure. In situ subcellular fractionation allows the visualization of protein compartmentalization and can also reveal protein sub-populations that localize to specific structures. For example, removal of soluble cytoplasmic proteins and loosely held nuclear proteins can reveal the stable association of some transcription factors with chromatin. Subsequent digestion of DNA can in some cases reveal association with the network of proteins and RNAs that is collectively termed the nuclear scaffold or nuclear matrix. 
Here we describe the steps required during the in situ fractionation of adherent and non-adherent mammalian cells on microscope coverslips. Protein visualization can be achieved using specific antibodies or fluorescent fusion proteins and fluorescence microscopy. Antibodies and/or fluorescent dyes that act as markers for specific compartments or structures allow protein localization to be mapped in detail. In situ fractionation can also be combined with western blotting to compare the amounts of protein present in each fraction. This simple biochemical approach can reveal associations that would otherwise remain undetected.

In situ Subcellular Fractionation of Adherent and Non-adherent Mammalian Cells

In situ subcellular fractionation of mammalian cells on microscope coverslips allows the visualisation of protein localisation.

Yapışkan ve Sigara yapışık Memeli Hücre yerinde Subselüler Fraksiyonu

Protein function is intimately coupled to protein localization. Although some proteins are restricted to a specific location or subcellular compartment, ...

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the environment, but also determines body shape and plays a role in motility4-6. Several layers secreted by epidermal cells comprise the cuticle, including an outermost lipid layer7.
Circumferential ridges in the cuticle called annuli pattern the length of the animal and are present during all stages of development8. Alae are longitudinal ridges that are present during specific stages of development, including L1, dauer, and adult stages2,9. Mutations in genes that affect cuticular collagen organization can alter cuticular structure and animal body morphology5,6,10,11. While cuticular imaging using compound microscopy with DIC optics is possible, current methods that highlight cuticular structures include fluorescent transgene expression12, antibody staining13, and electron microscopy1. Labeled wheat germ agglutinin (WGA) has also been used to visualize cuticular glycoproteins, but is limited in resolving finer cuticular structures14. Staining of cuticular surface using fluorescent dye has been observed, but never characterized in detail15. We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. This optimized protocol for DiI staining is a simple, robust method for high resolution fluorescent visualization of annuli, alae, vulva, male tail, and hermaphrodite tail spike in C. elegans.

Visualization of Caenorhabditis elegans Cuticular Structures Using the Lipophilic Vital Dye DiI

We present a method to visualize cuticle in live C. elegans using the red fluorescent lipophilic dye DiI (1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate), which is commonly used in C. elegans to visualize environmentally exposed neurons. With this optimized protocol, alae and annular cuticular structures are stained by DiI and observed using compound microscopy.

Görselleştirme Caenorhabditis Elegans Tırnak Yapıları Lipofilik Vital Boya, DII

The cuticle of C. elegans is a highly resistant structure that surrounds the exterior of the animal1-4. The cuticle not only protects the animal from the ...

Do-It-Yourself Device for Recovery of Cryopreserved Samples Accidentally Dropped into Cryogenic Storage Tanks

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term storage of biological materials such as blood, cells and tissues 1,2. The cryogenic nature of liquid nitrogen, while ideal for sample preservation, can cause rapid freezing of live tissues on contact - known as 'cryogenic burn'2, which may lead to severe frostbite in persons closely involved in storage and retrieval of samples from Dewars. Additionally, as liquid nitrogen evaporates it reduces the oxygen concentration in the air and might cause asphyxia, especially in confined spaces2.
In laboratories, biological samples are often stored in cryovials or cryoboxes stacked in stainless steel racks within the Dewar tanks1. These storage racks are provided with a long shaft to prevent boxes from slipping out from the racks and into the bottom of Dewars during routine handling. All too often, however, boxes or vials with precious samples slip out and sink to the bottom of liquid nitrogen filled tank. In such cases, samples could be tediously retrieved after transferring the liquid nitrogen into a spare container or discarding it. The boxes and vials can then be relatively safely recovered from emptied Dewar. However, the cryogenic nature of liquid nitrogen and its expansion rate makes sunken sample retrieval hazardous. It is commonly recommended by Safety Offices that sample retrieval be never carried out by a single person. Another alternative is to use commercially available cool grabbers or tongs to pull out the vials3. However, limited visibility within the dark liquid filled Dewars poses a major limitation in their use.
In this article, we describe the construction of a Cryotolerant DIY retrieval device, which makes sample retrieval from Dewar containing cryogenic fluids both safe and easy.

Here we present a low cost, durable cryotolerant device for sample retrieval from Dewar tanks filled with liquid nitrogen. The ease of construction and modular design of the device makes the process of sample retrieval from cryogenic tanks safe and easy.

KRİYOPREZERVE Numune Kurtarma için Do-It-Yourself Aygıt yanlışlıkla Kriyojenik Depolama Tankları içine Dropped

Liquid nitrogen is colorless, odorless, extremely cold (-196 &deg;C) liquid kept under pressure. It is commonly used as a cryogenic fluid for long term ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

Protein-Protein Etkileşimi Okumak Manyetik Boncuk Kullanarak Bir Sıvı Faz Affinity Yakalama Testi: Poliovirüs-Nanobody Örnek

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

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 ...

Polysome Fractionation and Analysis of Mammalian Translatomes on a Genome-wide Scale

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. Accordingly, dysregulation of mRNA translation is considered to play a major role in a variety of pathological states including cancer. Ribosomes also host chaperones, which facilitate folding of nascent polypeptides, thereby modulating function and stability of newly synthesized polypeptides. In addition, emerging data indicate that ribosomes serve as a platform for a repertoire of signaling molecules, which are implicated in a variety of post-translational modifications of newly synthesized polypeptides as they emerge from the ribosome, and/or components of translational machinery. Herein, a well-established method of ribosome fractionation using sucrose density gradient centrifugation is described. In conjunction with the in-house developed &#8220;anota&#8221; algorithm this method allows direct determination of differential translation of individual mRNAs on a genome-wide scale. Moreover, this versatile protocol can be used for a variety of biochemical studies aiming to dissect the function of ribosome-associated protein complexes, including those that play a central role in folding and degradation of newly synthesized polypeptides.

Ribosomes play a central role in protein synthesis. Polyribosome (polysome) fractionation by sucrose density gradient centrifugation allows direct determination of translation efficiencies of individual mRNAs on a genome-wide scale. In addition, this method can be used for biochemical analysis of ribosome- and polysome-associated factors such as chaperones and signaling molecules.

Bir genom Ölçeği Polizom Fraksiyonu ve Memeli Translatomes Analizi

mRNA translation plays a central role in the regulation of gene expression and represents the most energy consuming process in mammalian cells. ...

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we showed that D. discoideum is remarkably resilient to protein aggregation and can be used to gain insights into the cellular protein quality control system. However, the use of D. discoideum as a model system poses several challenges to microscopy-based experimental approaches, such as the high motility of the cells and their susceptibility to photo-toxicity. The latter proves to be especially challenging when studying protein homeostasis, as the phototoxic effects can induce a cellular stress response and thus alter to behavior of the protein quality control system. 
Temperature increase is a commonly used way to induce cellular stress. Here, we describe a temperature-controllable imaging protocol, which allows observing temperature-induced perturbations in D. discoideum. Moreover, when applied at normal growth temperature, this imaging protocol can also noticeably reduce photo-toxicity, thus allowing imaging with higher intensities. This can be particularly useful when imaging proteins with very low expression levels. Moreover, the high mobility of the cells often requires the acquisition of multiple fields of view to follow individual cells, and the number of fields needs to be balanced against the desired time interval and exposure time.

Studying the Protein Quality Control System of D. discoideum Using Temperature-controlled Live Cell Imaging

The social amoebae Dictyostelium discoideum has recently been established as a system to study protein misfolding and proteostasis. Here, we describe a new imaging-based methodology to study temperature-induced protein aggregation and the cellular stress response in D. discoideum.

Protein Kalite Kontrol Sistemi incelenmesi<em&gt; D. diskodeyum</em&gt; Sıcaklık kontrollü Canlı Hücre Görüntüleme kullanarak

The complex lifestyle of the social amoebae Dictyostelium discoideum makes it a valuable model for the study of various biological processes. Recently, we ...

Subcellular Fractionation for ERK Activation Upon Mitochondrial-derived Peptide Treatment

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the mitochondrial genome. MDPs have a wide variety of biological effects such as protecting neurons from apoptosis, improving metabolic markers, and protecting cells from chemotherapy. Humanin was the first MDP to be discovered and is the most studied peptide among the MDP family. The membrane receptors and downstream signaling pathways of humanin have been carefully characterized. Additional MDPs such as MOTS-c and SHLP1-6 have been more recently discovered and the signaling mechanisms have yet to be elucidated. Here we describe a cell culture based method to determine the function of these peptides. In particular, cell fractionation techniques in combination with western blotting allow for the quantitative determination of activation and translocation of important signaling molecule. While there are other methods of cell fractionation, the one described here is an easy and straightforward method. These methods can be used to further elucidate the mechanism of action of these peptides and other therapeutic agents.

This protocol describes how to stimulate cells with mitochondrial-derived peptides and assess the signaling cascade and localization of phospho-proteins.

Mitokondrial kaynaklı peptid tedavi üzerine ERK harekete geçirmek için hücre altı ayırma

Mitochondrial-derived peptides (MDPs) are a new class of peptides that are encoded by small open reading frames within other known genes of the ...

Graphene Enclosure of Chemically Fixed Mammalian Cells for Liquid-Phase Electron Microscopy

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using scanning transmission electron microscopy (STEM). Cells of the mammalian breast cancer cell line SKBR3 were grown on silicon microchips with silicon nitride (SiN) windows. Cells were chemically fixed, and HER2 proteins were labeled with quantum dot nanoparticles (QDs), using a two-step biotin-streptavidin binding protocol. The cells were coated with multilayer graphene to maintain a hydrated state, and to protect them from electron beam damage during STEM. To examine the stability of the samples under electron beam irradiation, a dose series experiment was performed. Graphene-coated and non-coated samples were compared. Beam induced damage, in the form of bright artifacts, appeared for some non-coated samples at increased electron dose D, while no artifacts appeared on coated samples.

Presented here is a protocol for labeling membrane proteins in mammalian cells and coating the sample with graphene for liquid-phase scanning transmission electron microscopy. The stability of the samples against the damage caused by radiation can also studied with this protocol.

Sıvı Fazlı Elektron Mikroskobu için Kimyasal Olarak Sabit Memeli Hücrelerinin Grafen Muhafazası

A protocol is described for investigating the human epidermal growth factor receptor 2 (HER2) in the intact plasma membrane of breast cancer cells using ...