1. 샘플 단백질 준비 <ol><li> 500 μl microfuge 관에서 다음을 결합 : </li></ol><table border="1"><tbody><tr><td> 시약 </td><td> : 추가 볼륨 </td></tr><tr><td> * 단백질 샘플 (최대 1 MG) </td><td> 최대 112 μL에 </td></tr><tr><td> 카트리지 키트에 제공된 샘플 버퍼 (5 ​​배) </td><td> 30 μL </td></tr><tr><td> DTT 1M </td><td> 8μL </td></tr><tr><td> 18 Ω H 2 O </td><td> X 150μL의 샘플 볼륨의 총 μL이 ** </td></tr></tbody></table><ol start="2"><li> ° C 5 분 95에서 샘플을 변성. 실내 온도 쿨. </li></ol> * 분리되는 단백질의 총 금액은 샘플의 복잡성에 따라 달라집니다. 이러한 조직의 homogenate 같은 좀 더 복잡한 샘플, 들어, 채널 당 150-200 μg을 권장합니다. 샘플 볼륨 미만 112 μL입니다 ** 경우, 150 μL에 최종 볼륨을 가지고 18 Ω H 2 O를 추가할 수 있습니다. 2. GELFREE 카트리지를 로딩 <ol><li> 호일 주머니에서 GELFREE 8100 카트리지를 제거합니다. </li><li> 제거하고 판 실러를 폐기하십시오. </li><li> 피펫을 사용하여 카트리지 구획에서 저장 버퍼를 제거합니다. 카트리지의 모든 8 실이 사용되는 경우, 카트리지는 구획에서 저장 버퍼를 유출하는 반전 수 있습니다. </li><li> 다음과 같이 카트리지 실 각 GELFREE 실행 버퍼를 추가하여 로딩을 위해 카트리지를 준비 : </li></ol><table border="1"><tbody><tr><td> GELFREE 카트리지 챔버 </td><td> 추가 GELFREE 실행 버퍼의 볼륨 </td></tr><tr><td> 양극 버퍼 저수지 </td><td> 8 ML </td></tr><tr><td> 음극 버퍼 저수지 </td><td> 6 ML </td></tr><tr><td> 콜렉션 챔버스 </td><td> 100 μL 각 </td></tr></tbody></table><ol start="5"><li> 샘플 로딩 챔버로부터 버퍼를 제거하고 폐기. </li><li> 8 채널 pipettor를 사용하여 로딩 챔버에 150 μL 샘플을로드합니다. </li><li> GELFREE 8100 분획화 역에 로드된 카트리지를 놓습니다. 전극 배열을 낮추고 뚜껑을 닫습니다. </li></ol> 3. 카트리지를 실행 <ol><li> 악기의 터치 스크린 디스플레이를 사용하여 미리 프로그램된 방법의 목록을 보려면 방법 버튼을 누르십시오. 미리 프로그램된 방법 각각의 분자 무게의 특정 범위 내에서 단백질을 해결하기위한 설정 전압 및 시간 기반 일시 중지로 구성되어 있습니다. GELFREE 8100는 방법에 지정된 간격으로 분수 수집에 대해 자동으로 일시 정지됩니다. </li><li> 적절한 방법이 발견되면 검색 버튼을 누르면, 화면의 키패드를 사용하여 원하는 미리 프로그램된 방식의 번호를 입력합니다. 확인을 누르면 다음 적용 버튼을 누릅니다. 적용되고있는 방법은 메인 화면의 하단에 나타납니다. </li><li> 실행에 사용됩니다 채널 (샘플 번호)를 선택하려면 주 화면에서 채널 버튼을 누르십시오. 몽땅 채널을 선택하려면 모두 선택을 누릅니다. 그런 다음, 기본 화면으로 돌아갑니다 완료 누르십시오. </li><li> 안전 덮개가 닫혀 있는지 확인하고 표시등이 화면 하단에 녹색입니다. </li><li> 단백질 분리를 시작하려면 시작을 누르십시오. . 그것이 분수 참고를 수집하는 시간이되면 분획화 스테이션 일시 자동 : 시스템이 실행되는 동안, 화면의 왼쪽에있는 상자에 표시된 동그라미는 각 채널의 상태를 묘사. 녹색 동그라미는 채널이 활성 상태임을 나타냅니다, 노란색 원은 악기 일시 중지했음을 나타냅니다, 그리고 빨간색 동그라미 채널이 실패했음을 나타냅니다. 적용 전압 및 전류는 채널 상태 동아리의 오른쪽에 표시됩니다. 상태 원에 노란색 설정되어 있고 텍스트 경고가 악기의 화면에 나타날 때 분수는 제거되어야합니다. </li><li> 분수를 제거하려면, 악기의 뚜껑을 열고 수집 실의 각 150 μL를 철회하는 8 채널 피펫을 사용합니다. </li><li> 분수가 제거되고 나면, 채널 당 100 μl를 추가하고 두 번하고 위아래로 pipetting하여 GELFREE 실행 버퍼와 함께 두 번 수집 챔버를 세척. </li><li> 세척 후, 수집 실에 다시 버퍼를 실행 GELFREE의 100uL를 추가 뚜껑을 닫은 이력서 버튼을 누르십시오. </li><li> GELFREE 8100은 다음 시간 간격까지 실행됩니다. 실험에 남아있는 총 시간은 화면의 오른쪽에 표시됩니다. 남은 시간 사이를 전환하려면 시간이 경과하고, 일시 정지 시간, 시간을 표시 상자 위에있는 버튼을 누르십시오. </li><li> 일단 분수의 모든 공동되었습니다llected는, 그들은 즉시 isoelectric 초점이나 겔 전기 영동으로 더 하류 준비, 또는 액체 크로마 토그래피 질량 분석계 또는 면역 친 화성을 사용하여 분석을 위해 사용될 수 있습니다. </li></ol> 4. 대표 결과 보빈 간 homogenate의 200 μg가 GELFREE 분류 시스템을 사용하여, 12 분수 각으로 분할되었다. 각 분획 6 μL는 표준 1D SDS 페이지 젤과 실버 스테인드에서 실행되었습니다. 그림 1에 나타난 단백질 샘플 3.5kDa에서 150kDa으로 분자량 범위를 스팬, 12 독특한 분수로 fractionated되었습니다. 분자량 표준은 (ColorBurst, 시그마)도 차선 하나 14에 표시됩니다. 여기에 표시된 겔은 단백질 분류에 대한 GELFREE 8100 시스템을 사용하여 달성 높은 복구 속도를 강조 표시합니다. 여기에 표시된 젤 200 μg에서 발견한 단백질의 4 %를 나타냅니다. <img src="/files/ftp_upload/1842/1842fig1tmb.jpg" alt="그림 1" border="0" /> 그림 1. 보빈 간장에서 단백질은 GELFREE 분류 시스템을 사용하여 분리 homogenate. 각 150 μL 분수 6 μL가로드 및 1D SDS 페이지 젤과 실버 스테인드에서 실행되었습니다. 표시된으로 ​​분자량 (MW) 표준도로드되었다. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1842/1842fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다.

<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>

이 문서의 저자는이 문서에서 사용되는 시약 및 악기를 생산하는 단백질 발견으로 고용됩니다.

GELFREE 8100 분획화 시스템은 이산 분자량 기반의 분수로 복잡한 단백질 시료를 파티션을위한 빠르고, 간단하고 재현성 방법을 제공합니다. 분류의 방법이 소설은 가능한 90 분 이내에 광범위한 질량 범위 단백질을 분리하고, 밴드 또는 현장 절단에 대한 필요가 없습니다. 샘플 비교적 대량로드하고 액상의 분수를 복구할 수있는 능력은 일반적으로 검출에 더 어렵습니다 낮은 풍부한 단백질의 분리 및 분석에 특히 유용합니다. 그 결과 분수는 다음과 같은 LC - MS, 든 - TOF 및 immunoblotting 같은 어플 리케이션의 다양한 배열에서 사용할 수 있습니다.

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

GELFREE 8100 분획화 시스템은 분자량을 기반으로 분류하는 동안 단백질 복구를 극대화하기 위해 설계된 새로운 단백질 분획화 시스템입니다. 시스템은 단일 사용, 8 샘플 용량 카트리지와 benchtop GELFREE 분별 (분리) 악기로 구성되어 있습니다. 분리하는 동안 일정한 전압은 양극과 음극의 저수지 사이에 적용되며, 각각의 단백질 혼합물은 electrophoretically 로딩 챔버에서 특별히 고안된 젤 열 젤로 구동됩니다. 단백질은 스태킹 젤에 꽉 밴드에 집중하고, 해결 젤에 해당 전기 mobilities에 따라 구분됩니다. 단백질은 칼럼에서 elute으로, 그들은 수집 챔버에서 젤 무료 갇혀 액상에 집중되어있다. 악기 그런 다음 특정 시간 간격을 일시 중지하고, 분수는 피펫을 사용하여 수집하고 있습니다. 모든 원하는 분수가 수집 때까지이 과정이 반복됩니다. 이하 8 샘플 카트리지에서 실행하는 경우, 사용하지 않은 챔버 스는 후속 분리에 사용할 수 있습니다. 이 소설의 기술은 동시에 8 복잡한 단백질 혼합물까지 빠르고 간단한 분리를 용이하게하고, 이전에 사용 가능한 분류 방법에 비해 몇 가지 장점을 제공합니다. 이 시스템은 카트리지 당 8mg 총, 채널 당 총 단백질 1mg까지 fractionating이 가능합니다. 넓은 질량 범위에서 그대로 단백질은 analyte 중요한 physiochemical 속성을 유지, 분자량을 바탕으로 구분됩니다. 1 분획화 프로세스가 높은 재현성 만들기, 밴드 또는 현장 절단에 대한 필요성을 제거하면서 액상의 함정 수사가 높은 복구를 위해 제공합니다.

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

함께 비디오 GELFREE 8100 분획화 시스템의 사용, 분자량을 기준으로 파티션 복잡한 단백질 샘플을 설명하고 액상에있는 분수를 복구합니다. 비디오 기술이 사용 방법, 작동 및 fractionated 소 간 homogenate의 polyacrylamide 젤 전기 영동 분석, 결과 데이터를 제공하는 방법에 대해 설명합니다.

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

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 조회수.  Protein Discovery, Inc.. 함께 비디오 GELFREE 8100 분획화 시스템의 사용, 분자량을 기준으로 파티션 복잡한 단백질 샘플을 설명하고 액상에있는 분수를 복구합니다. 비디오 기술이 사용 방법, 작동 및 fractionated 소 간 homogenate의 polyacrylamide 젤 전기 영동 분석, 결과 데이터를 제공하는 방법에 대해 설명합니다.

비디오: 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 에그의 작성과 분류는 압축을 풉니다

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

연구

생물학

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.

자기편 및 비 점착 성의 포유 동물 세포의 현장 Subcellular 분별 (분리)에서

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.

의 시각화 Caenorhabditis Elegans</emLipophilic 바이탈 염료, DiI를 사용하기> 표피 구조

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.

Cryopreserved 샘플 복구를 위해 손수 함 장치 실수로 극저온 저장 탱크 떨어졌고

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.

단백질 - 단백질 상호 작용 연구를 자성 비즈를 사용하여 액상 동질 캡처 분석 : Poliovirus-Nanobody의 예

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.

비교 단백체 분석을위한 신진 대사 라벨링 및 막 분별<em&gt; 애기 장대</em&gt; 서스펜션 세포 배양

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.

게놈 전체 규모에 Polysom​​e 분별과 포유류 Translatomes 분석

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.

단백질의 품질 관리 시스템을 공부<em&gt; D. discoideum</em&gt; 온도 제어 라이브 셀 이미징을 사용하여

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.

미토 콘 드리 아에서 파생 된 펩 티 드 치료 시 ERK 활성화에 대 한 subcellular 분류

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.

액체 상 전자 현미경 검사를 위한 화학적으로 고정된 포유류 세포의 그래핀 인클로저

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