우리는 refolding 조건을 최적화에 대한 도움 박사 Naidenko 감사합니다. 이 작품은 로랑 Brossay에 NIH 연구 부여 AI58181에 의해 지원되었다. 신디 반은 NIH NRSA F31 AI080230에 의해 지원됩니다. 스테파니 Terrizzi와 신디 반이 작품에 동등하게 기여하고 있습니다.

포함 기관의 준비 <ol><li> TB 4 X 500 ML flasks의 예방 각각 KLRG1 플라스미드 구조를 표현하는 박테리아의 야간 문화와 50 μg / carbenicillin과 chloramphenicol의 ML을 포함. OD 600 nm의에서 0.6 A를 도달하면, 37에서 0.4 M IPTG의 추가와 함께 유도하고 떨고 추가 4 시간 동안 문화를 품어 ° C. </li><li> resuspension 버퍼 산도에서 수확한 세포를 Resuspend = 8.0 (50 MM 트리스 - HCL, 25 % (W / V) 자당, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 10 MM DTT). 참고 : 알약이 단계에서 냉동 수 있습니다. </li><li> 폴리 프로필렌의 비커에 세균성 resuspensions의 더 이상 60 이상 ML을 풀. 필요는 TE 버퍼 산도 8.0 60 ML에 적응 절반 속도로 혼합물을 저어하는 경우. </li><li> 혼합물을 교반하려면 드롭 현명한 추가 라이 소 자임 (최종 = 1 MG / ML), MgCl2 (최종 = 5 ㎜), 2 MG DNAse 나는 50 % 글리세롤 75 MM NaCl, 트리톤 - X100 (최종 = 1 %), DTT를 (들어 최종 = 10 ㎜). </li><li> 1.5 분 얼음 솔루션을 Sonicate 4에서 10 분, 5,000 RPM에서 lysates을 원심 ° C. <ol class="lalpha"><li> 뜨는을 가만히 따르다. </li><li> 세척 버퍼 산도 15 ML 추가 = 8.0 (50 MM 트리스 - HCL, 0.5 % 트리톤 X - 100, 100 MM NaCl, 1 MM EDTA (에틸렌 다이아 민 테트라 초산), 1 MM DTT). </li><li> 펠렛가 완전히 resuspended 때까지 얼음, 1.5 분 솔루션을 sonicate. </li><li> 4 10 분 5,000 RPM에서 샘플을 원심 ° C. </li><li> 세탁 5 회 반복합니다. </li></ol></li><li> TE 버퍼 4 ML에서 트리톤 X - 100, 원심 분리기 및 resuspend없이 세척 버퍼와 5B를 반복합니다. </li><li> 30-100 MG / ML의 농도에 젖은 무게 포함 기관의 슬러리 및 TE 버퍼에 저장을 가져가라. </li></ol> KLRG1의 Refolding <ol><li> 버퍼 산도를 refolding 1 L 만들기 = 8.0 (0.4 M 아르기닌 - HCL, 0.1 M 트리스 산도 8-8.3, 2 MM EDTA (에틸렌 다이아 민 테트라 초산), 0.2 MM PMSF, 3 EDTA (에틸렌 다이아 민 테트라 초산) - 무료 테아제 억제제의 알약, 5 MM의 GSH와 0.5 MM의 GSSG)과 오한 4 ° C 감동하면서. </li><li> 접는 혼합물에 주입에 대한 포함 시체를 준비 : 이것은 단백질의 최종 농도가 2 3 μm의 될 수 있도록 0.25 0.5 μm의 농도로 각 5 10 주사를 만들기 위해 이상적입니다. <ol class="lalpha"><li> 10 MM β - 메르 캅 토 에탄올과 7 M GnHCl에 포함 기관 슬러리의 50 MG의 볼륨을 용융. </li><li> 유지 37 포함 기관 / GnHCl 혼합 ° 30 C - 40 분 및 와동 매 5 분 (슬러리가 완전히 녹아되면 취소가됩니다) 전체 융해를 보장합니다. </li><li> 4 microcentrifuge 30 분 최대 속도로 원심 분리기 ° C와 15 ML의 원심 관에 뜨는을 전송하기만하면됩니다. 작은 거무스름한 펠릿 중 하나를 전송하지 마십시오. </li></ol></li><li> 사출 버퍼 (3 M GnHCl, 10 MM의 NaAcetate, 산도 4.2,10 MM EDTA (에틸렌 다이아 민 테트라 초산))로 볼륨을 조정하십시오. </li><li> 희석 포함 단체 1 ML 매 시간마다 주사. 주입하면, 높은 속도로 저어 각 드롭 사이 이초와 드롭하여 희석 포함 단체 드롭 1 ML를 추가합니다. 주입이 완료되면 낮은 속도로 저어. </li><li> 4 낮은 속도로 감동 밤새 계속 ° C. </li></ol> Refolding 반응 농도 <ol><li> Millipore의 프로토콜에 따라, Amicon를 사용하여 사전에 필터링 (0.22 μm의이 여과) refolding 반응의 제 1 L를 집중 셀 및 ~ 10 ML에 YM10 NMWL 10K의 ultrafiltration 막 (Millipore)를 만들어 냈다. </li><li> 사용 Centriplus YM - 10 10K MWCO (Millipore).을 ≤ 2 ML에 집중 </li></ol> KLRG1 tetramer의 정화 <ol><li> 4 AKTAFPLC를 설정 ° C GE 헬스케어 설명서에 따라. </li><li> Sephacryl S - 200 60분의 16 고해상도 20 MM의 HEPES에서 열 (GE 헬스케어) 150 MM NaCl을 사용하여 크기를 제외 크로마 토그래피에 의해 KLRG1의 모노머 양식을 정화. (그림 1 참조). </li><li> Centriplus를 사용하여 한 ML에 집중 YM - 10 10K MWCO (Millipore). </li><li> KLRG1 단량체를 biotinylate하는 욕망의 프로토콜에 따라 비라 효소를 사용합니다. </li><li> 무료 비오틴은 2와 같은 크기 배제 크로마 토그래피에 의해 제거됩니다. </li><li> KLRG1 분자는 4 배 초과 어금니 PE 복합 streptavidin (BD Biosciences)와 KLRG1 단량체를 혼합하여 tetramerized 있습니다. </li></ol> 대표 결과 <a href="https://www-jove-com.remotexs.ntu.edu.sg/files/ftp_upload/1702/1702fig1.jpg"><img src="/files/ftp_upload/1702/1702fig1tmb.jpg" alt="그림 1" border="0" /></a> 의 악타 FPLC 크기 배제 크로마 토그래피에서 그림 1. 대표 긍정적인 결과 KLRG1를 refolded. 그래프는 단량체의 분리 (83.52 ML), 이합체 (56.36 ML) 및 refolded KLRG1의 multimer (36.26 ML) 양식을 보여줍니다. 94.25 ML에있는 정상은 버퍼 교환을 나타냅니다.

<ol>
	<li>Tessmer, M. S., Fugere, C., Stevenaert, F., Naidenko, O. V., Chong, H. J., Leclercq, G., Brossay, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=KLRG1+binds+cadherins+and+preferentially+associates+with+SHIP-1.">KLRG1 binds cadherins and preferentially associates with SHIP-1.</a> Int Immunol. 19, 391-400 (2007).</li><li>Grundemann, C., Bauer, M., Schweier, O., von Oppen, N., Lassing, U., Saudan, P., Becker, K. F., Karp, K., Hanke, T., Bachmann, M. F., Pircher, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cutting+edge:+identification+of+E-cadherin+as+a+ligand+for+the+murine+killer+cell+lectin-like+receptor+G1.">Cutting edge: identification of E-cadherin as a ligand for the murine killer cell lectin-like receptor G1.</a> J Immunol. 176, 1311-1315 (2006).</li><li>Ito, M., Maruyama, T., Saito, N., Koganei, S., Yamamoto, K., Matsumoto, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Killer+cell+lectin-like+receptor+G1+binds+three+members+of+the+classical+cadherin+family+to+inhibit+NK+cell+cytotoxicity.">Killer cell lectin-like receptor G1 binds three members of the classical cadherin family to inhibit NK cell cytotoxicity.</a> J Exp Med. 203, 289-295 (2006).</li><li>Li, Y., Hofmann, M., Wang, Q., Teng, L., Chlewicki, L. K., Pircher, H., Mariuzza, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structure+of+natural+killer+cell+receptor+KLRG1+bound+to+E-cadherin+reveals+basis+for+MHC-independent+missing+self+recognition.">Structure of natural killer cell receptor KLRG1 bound to E-cadherin reveals basis for MHC-independent missing self recognition.</a> Immunity. 31, 35-46 (2009).</li><li>Nakamura, S., Kuroki, K., Ohki, I., Sasaki, K., Kajikawa, M., Maruyama, T., Ito, M., Kameda, Y., Ikura, M., Yamamoto, K., Matsumoto, N., Maenaka, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=K.+Molecular+basis+for+E-cadherin+recognition+by+killer+cell+lectin-like+receptor+G1+(KLRG1).">K. Molecular basis for E-cadherin recognition by killer cell lectin-like receptor G1 (KLRG1).</a> J Biol Chem. , .</li></ol>

이 프로토콜에서는, 그것은 biotinylate, 정화하는 방법을 표시하고, tetramerize KLRG1입니다. KLRG1 tetramer는 유동세포계측법로 KLRG1 리간드 레이블을 사용할 수 있습니다. refolding 조건이 각각의 단백질에 대해 경험적으로 테스트해야되지만, 다른 C - 타입 lectins은 유사한 프로토콜을 사용 tetramerized 수 있습니다. 프로브, 고아 C - 타입 렉틴 수용체에 리간드로 tetramerized 단백질을 사용하면 잠재적으로 식별 수 있습니다.

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		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
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<tr>
<td>BirA Enzyme and Buffer</td>
<td>Biotinylation Kit</td>
<td>Avidity</td>
<td>BIRA500</td>
<td>&nbsp;</td>
<tr>
<td>Complete Mini Protease Inhibitor Tablets, EDTA Free</td>
<td>Reagent</td>
<td>Roche</td>
<td>11 836 170 001</td>
<td>or equivalent</td>
</tr>
<tr>
<td>Amicon Stirred Cell</td>
<td>Equipment</td>
<td>Millipore</td>
<td>Model #8400</td>
<td>or equivalent</td>
</tr>
<tr>
<td>YM10 NMWL 10K ultrafiltration membrane</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>13642</td>
<td>&nbsp;</td>
<tr>
<td>15 ml YM10 concentrator</td>
<td>Filtration Supply</td>
<td>Millipore</td>
<td>4411</td>
<td>&nbsp;</td>
<tr>
<td>AKTAFPLC</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>18-1900-26</td>
<td>&nbsp;</td>
<tr>
<td>Sephacryl S-200 16/60 high-resolution column</td>
<td>Equipment</td>
<td>GE Healthcare</td>
<td>17-1166-01</td>
<td>&nbsp;</td>
<tr>
<td>Strepavidin PE</td>
<td>Reagent</td>
<td>BD Biosciences</td>
<td>554061</td>
<td>or equivalent</td>
</tr>		</tbody>
		</table>
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a protocol for the production of klrg1 tetramer

킬러 세포 렉틴 같은 수용체 G1 (KLRG1)은 C 타입 렉틴 같은 superfamily에 속하는 유형 II의 transmembrane의 당단백질 수용체 억제합니다. KLRG1는 단량체로서 이황화 결합 homodimer로 모두 존재합니다. 이 잘 보존되어 수용체가 가장 성숙하고 최근에 활성화된 NK 세포에서뿐만 아니라 효과기 / 메모리 T 세포의 일부에서 발견됩니다. KLRG1 tetramer뿐만 아니라 다른 방법을 사용하여, E -, N - 및 R - cadherins은 KLRG1 리간드로 확인되었습니다. 이러한 칼슘 2 +에 의존 세포 - 세포 부착 분자는 세포 - 세포 상호 작용, transmembrane 도메인과 굴지의 cytoskeleton에 연결된 세포질 도메인에 대해 책임을 다섯 cadherin의 반복을 포함하는 세포외 도메인에 구성되어 있습니다. KLRG1 tetramer의 생성은 KLRG1 리간드의 식별에 필수되었습니다. KLRG1 tetramer 또한 면역 반응 및 조직 무결성을 규제의 역할 cadherin과 KLRG1 재생을 명료하게하다 수있는 유일한 도구입니다.

Watch this Scientific Journal Video about A Protocol for the Production of KLRG1 Tetramer at JoVE.com

A Protocol for the Production of KLRG1 Tetramer

이 프로토콜은 KLRG1 리간드의 분석을위한 강력한 도구입니다 KLRG1 tetramer의 생산을 설명합니다.

KLRG1 Tetramer의 생산을위한 프로토콜

Killer cell lectin-like receptor G1 (KLRG1) is a type II transmembrane glycoprotein inhibitory receptor belonging to the C type lectin-like superfamily. ...

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9.9K 조회수.  Brown University. 이 프로토콜은 KLRG1 리간드의 분석을위한 강력한 도구입니다 KLRG1 tetramer의 생산을 설명합니다.

비디오: A Protocol for the Production of KLRG1 Tetramer

Protocol for Mosquito Rearing (A. gambiae)

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night cycle. For this procedure, you'll need mosquito cages, 10% sterile sucrose solution, paper towels, beaker, whatman filter paper, glass feeders, human blood and serum, water bath, parafilm, distilled water, clean plastic trays, mosquito food (described below), mosquito net to cover the trays, vacuum, and a collection chamber to collect adults.

Protocol for Mosquito Rearing A. gambiae

This video illustrates the general techniques used to rear Anopheles gambiae in the laboratory. The methods for caring for laboratory mosquitoes are demonstrated through all stages of the organism's life cycle from larvae to pupae to blood-feeding adults.

모스키토 리어링 (A. gambiae)에 대한 프로토콜

This protocol describes mosquito rearing in the insectary. The insectary rooms are maintained at 28&deg;C and ~80% humidity, with a 12 hr. day/night ...

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Visualizing Antigen Specific CD4+ T Cells using MHC Class II Tetramers

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method relies on the highly specific interaction between peptide loaded MHC and the corresponding T-cell receptor.  While the affinity of a single MHC/peptide molecule is low, cross-linking MHC/peptide complexes with streptavidin increases the avidity of the interaction, enabling their use as staining reagents.  Because of the relatively low frequencies of CD4+ T cells (~1 in 300,000 for a single specificity) this assay utilizes an in vitro amplification step to increase its threshold of detection.  Mononuclear cells are purified from peripheral blood by Ficoll underlay.  CD4+ cells are then separated by negative selection using biotinylated antibody cocktail and anti-biotin labeled magnetic beads.  Using adherent cells from the CD4- cell fraction as antigen presenting cells, CD4+ T cells are expanded in media by adding an antigenic peptide and IL-2.  The expanded cells are stained with the corresponding class II tetramer by incubating at 37 C for one hour and subsequently stained using surface antibodies such as anti-CD4, anti-CD3, and anti-CD25.  After labeling, the cells can be directly analyzed by flow cytometry.  The tetramer positive cells typically form a distinct population among the expanded CD4+ cells.  Tetramer positive cells are usually CD25+ and often CD4 high.  Because the level of background tetramer staining can vary, positive staining results should always be compared to the staining of the same cells with an irrelevant tetramer.  Multiple variations of this basic assay are possible.  Tetramer positive cells may be sorted for further phenotypic analysis, inclusion in ELISPOT or proliferation assays, or other secondary assays.  Several groups have also demonstrated co-staining using tetramers and either anti-cytokine or anti-FoxP3 antibodies.  

This procedure demonstrates the purification and in vitro expansion of antigen specific CD4+ T cells from whole peripheral blood and their visualization using MHC class II tetramers. Tetramers permit the direct visualization of T cells with a single antigen specificity and defined MHC class II restriction.

머릿속 항원 특정 CD4 + MHC 클래스 II Tetramers를 사용하여 T 세포

Major histocompatibility complex (MHC) class II tetramers allow the direct visualization of antigen specific CD4+ T cells by flow cytometry.  This method ...

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and expression pattern of genes of interest or generation of tandem affinity purification (TAP) tagged versions of specific genes to facilitate their purification. Typically transgenes are generated by placing a promoter upstream of a GFP reporter gene or cDNA of interest, and this often produces a representative expression pattern. However, critical elements of gene regulation, such as control elements in the 3' untranslated region or alternative promoters, could be missed by this approach. Further only a single splice variant can be usually studied by this means. In contrast, the use of worm genomic DNA carried by fosmid DNA clones likely includes most if not all elements involved in gene regulation in vivo which permits the greater ability to capture the genuine expression pattern and timing. To facilitate the generation of transgenes using fosmid DNA, we describe an E. coli based recombineering procedure to insert GFP, a TAP-tag, or other sequences of interest into any location in the gene. The procedure uses the galK gene as the selection marker for both the positive and negative selection steps in recombineering which results in obtaining the desired modification with high efficiency. Further, plasmids containing the galK gene flanked by homology arms to commonly used GFP and TAP fusion genes are available which reduce the cost of oligos by 50% when generating a GFP or TAP fusion protein. These plasmids use the R6K replication origin which precludes the need for extensive PCR product purification. Finally, we also demonstrate a technique to integrate the unc-119 marker on to the fosmid backbone which allows the fosmid to be directly injected or bombarded into worms to generate transgenic animals. This video demonstrates the procedures involved in generating a transgene via recombineering using this method.

The Production of C. elegans Transgenes via Recombineering with the galK Selectable Marker

The ability to produce transgenes for Caenorhabditis elegans using genomic DNA carried by fosmids is particularly attractive as all of the native regulatory elements are retained. Described is a simple and robust procedure for the production of transgenes via recombineering with the galK selectable marker.

생산 C. 엘레간스</em함께 Recombineering 통해> Transgenes galK 선택 마커

The creation of transgenic animals is widely utilized in C. elegans research including the use of GFP fusion proteins to study the regulation and ...

DNA Extraction from Paraffin Embedded Material for Genetic and Epigenetic Analyses

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number gains and losses and DNA methylation. Advances in high-throughput, genome-wide profiling technologies, such as microarrays, have significantly improved our ability to identify and detect these specific alterations. However as technology continues to improve, a limiting factor remains sample quality and availability. Furthermore, follow-up clinical information and disease outcome are often collected years after the initial specimen collection. Specimens, typically formalin-fixed and paraffin embedded (FFPE), are stored in hospital archives for years to decades. DNA can be efficiently and effectively recovered from paraffin-embedded specimens if the appropriate method of extraction is applied. High quality DNA extracted from properly preserved and stored specimens can support quantitative assays for comparisons of normal and diseased tissues and generation of genetic and epigenetic signatures 1. To extract DNA from paraffin-embedded samples, tissue cores or microdissected tissue are subjected to xylene treatment, which dissolves the paraffin from the tissue, and then rehydrated using a series of ethanol washes. Proteins and harmful enzymes such as nucleases are subsequently digested by proteinase K. The addition of lysis buffer, which contains denaturing agents such as sodium dodecyl sulfate (SDS), facilitates digestion 2. Nucleic acids are purified from the tissue lysate using buffer-saturated phenol and high speed centrifugation which generates a biphasic solution. DNA and RNA remain in the upper aqueous phase, while proteins, lipids and polysaccharides are sequestered in the inter- and organic-phases respectively. Retention of the aqueous phase and repeated phenol extractions generates a clean sample. Following phenol extractions, RNase A is added to eliminate contaminating RNA. Additional phenol extractions following incubation with RNase A are used to remove any remaining enzyme. The addition of sodium acetate and isopropanol precipitates DNA, and high speed centrifugation is used to pellet the DNA and facilitate isopropanol removal. Excess salts carried over from precipitation can interfere with subsequent enzymatic assays, but can be removed from the DNA by washing with 70% ethanol, followed by centrifugation to re-pellet the DNA 3. DNA is re-suspended in distilled water or the buffer of choice, quantified and stored at -20&deg;C. Purified DNA can subsequently be used in downstream applications which include, but are not limited to, PCR, array comparative genomic hybridization 4 (array CGH), methylated DNA Immunoprecipitation (MeDIP) and sequencing, allowing for an integrative analysis of tissue/tumor samples.

This video demonstrates the protocol for DNA extraction from formalin-fixed paraffin-embedded material. This is a multi-day procedure in which tissue sections are deparaffinized with xylene, rehydrated with ethanol and treated with proteinase K to purify and isolate DNA for subsequent gene-specific or genome-wide analysis.

유전과 Epigenetic 분석을위한 파라핀 임베디드 재료에서 DNA 추출

Disease development and progression are characterized by frequent genetic and epigenetic aberrations including chromosomal rearrangements, copy number ...

A Convenient and General Expression Platform for the Production of Secreted Proteins from Human Cells

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. However, prokaryotic hosts are often not as appropriate for expression of human, viral or eukaryotic proteins due to toxicity of the foreign macromolecule, differences in the protein folding machinery, or due to the lack of particular co- or post-translational modifications in bacteria. Expression systems based on yeast (P. pastoris or S. cerevisiae) 1,2, baculovirus-infected insect (S. frugiperda or T. ni) cells 3, and cell-free in vitro translation systems 2,4 have been successfully used to produce mammalian proteins. Intuitively, the best match is to use a mammalian host to ensure the production of recombinant proteins that contain the proper post-translational modifications. A number of mammalian cell lines (Human Embryonic Kidney (HEK) 293, CV-1 cells in Origin carrying the SV40 larget T-antigen (COS), Chinese Hamster Ovary (CHO), and others) have been successfully utilized to overexpress milligram quantities of a number of human proteins 5-9. However, the advantages of using mammalian cells are often countered by higher costs, requirement of specialized laboratory equipment, lower protein yields, and lengthy times to develop stable expression cell lines. Increasing yield and producing proteins faster, while keeping costs low, are major factors for many academic and commercial laboratories.
Here, we describe a time- and cost-efficient, two-part procedure for the expression of secreted human proteins from adherent HEK 293T cells. This system is capable of producing microgram to milligram quantities of functional protein for structural, biophysical and biochemical studies. The first part, multiple constructs of the gene of interest are produced in parallel and transiently transfected into adherent HEK 293T cells in small scale. The detection and analysis of recombinant protein secreted into the cell culture medium is performed by western blot analysis using commercially available antibodies directed against a vector-encoded protein purification tag. Subsequently, suitable constructs for large-scale protein production are transiently transfected using polyethyleneimine (PEI) in 10-layer cell factories. Proteins secreted into litre-volumes of conditioned medium are concentrated into manageable amounts using tangential flow filtration, followed by purification by anti-HA affinity chromatography. The utility of this platform is proven by its ability to express milligram quantities of cytokines, cytokine receptors, cell surface receptors, intrinsic restriction factors, and viral glycoproteins. This method was also successfully used in the structural determination of the trimeric ebolavirus glycoprotein 5,10.
In conclusion, this platform offers ease of use, speed and scalability while maximizing protein quality and functionality. Moreover, no additional equipment, other than a standard humidified CO2 incubator, is required. This procedure may be rapidly expanded to systems of greater complexity, such as co-expression of protein complexes, antigens and antibodies, production of virus-like particles for vaccines, or production of adenoviruses or lentiviruses for transduction of difficult cell lines.

In the post-human genomics era, the availability of recombinant proteins in native conformations is crucial to structural, functional and therapeutic research and development. Here, we describe a test- and large-scale protein expression system in human embryonic kidney 293T cells that can be used to produce a variety of recombinant proteins.

인간의 세포에서 분비 단백질의 생산을위한 편리하고 일반적인 표현 플랫폼

Recombinant protein expression in bacteria, typically E. coli, has been the most successful strategy for milligram quantity expression of proteins. ...

A Simple Protocol for Extracting Hemocytes from Wild Caterpillars

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle 1. In larval stages of insects belonging to the orders of Lepidoptera (moths and butterflies) and Diptera (true flies), hemocytes are formed from the lymph gland (a specialized hematopoietic organ) or embryonic cells and can be carried through to the adult stage. Embryonic hemocytes are involved in cell migration during development and chemotaxis regulation during inflammation. They also take part in cell apoptosis and are essential for embryogenesis 2. Hemocytes mediate the cellular arm of the insect innate immune response that includes several functions, such as cell spreading, cell aggregation, formation of nodules, phagocytosis and encapsulation of foreign invaders 3. They are also responsible for orchestrating specific insect humoral defenses during infection, such as the production of antimicrobial peptides and other effector molecules 4, 5. Hemocyte morphology and function have mainly been studied in genetic or physiological insect models, including the fruit fly, Drosophila melanogaster 6, 7, the mosquitoes Aedes aegypti and Anopheles gambiae 8, 9 and the tobacco hornworm, Manduca sexta 10, 11. However, little information currently exists about the diversity, classification, morphology and function of hemocytes in non-model insect species, especially those collected from the wild 12.
Here we describe a simple and efficient protocol for extracting hemocytes from wild caterpillars. We use penultimate instar Lithacodes fasciola (yellow-shouldered slug moth) (Figure 1) and Euclea delphinii (spiny oak slug) caterpillars (Lepidoptera: Limacodidae) and show that sufficient volumes of hemolymph (insect blood) can be isolated and hemocyte numbers counted from individual larvae. This method can be used to efficiently study hemocyte types in these species as well as in other related lepidopteran caterpillars harvested from the field, or it can be readily combined with immunological assays designed to investigate hemocyte function following infection with microbial or parasitic organisms 13.

Insect hemocytes carry out many important functions, both immune and non-immune, throughout all stages of insect development. Our present knowledge of hemocyte types and function comes from studies on insect genetic models. Here, we present a method for extracting, quantifying and visualizing hemocytes from wild caterpillars.

야생 유충에서 Hemocytes을 추출하기위한 간단한 프로토콜

Insect hemocytes (equivalent to mammalian white blood cells) play an important role in several physiological processes throughout an insect's life cycle ...

SIVQ-LCM Protocol for the ArcturusXT Instrument

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an advanced, rapidly customizable laser dissection platform technology. In this report, we describe the integration of the image analysis software Spatially Invariant Vector Quantization (SIVQ) onto the ArcturusXT instrument. The ArcturusXT system contains both an infrared (IR) and ultraviolet (UV) laser, allowing for specific cell or large area dissections. The principal goal is to improve the speed, accuracy, and reproducibility of the laser dissection to increase sample throughput. This novel approach facilitates microdissection of both animal and human tissues in research and clinical workflows.

SIVQ-LCM is an innovative approach that harnesses a computer algorithm, Spatially Invariant Vector Quantization (SIVQ), to drive the laser capture microdissection (LCM) process. The SIVQ-LCM workflow greatly improves the speed and accuracy of microdissection, with applications in both the research and clinical settings.

ArcturusXT 악기 SIVQ-LCM 프로토콜

SIVQ-LCM is a new methodology that automates and streamlines the more traditional, user-dependent laser dissection process.&#160;It aims to create an ...

Measurement and Analysis of Extracellular Acid Production to Determine Glycolytic Rate

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both mitochondrial (respiration) and non-mitochondrial (other redox) reactions consume oxygen, but these reactions can be easily distinguished by chemical inhibition of mitochondrial respiration. However, while mitochondrial oxygen consumption is an unambiguous and direct measurement of respiration rate2, the same is not true for extracellular acid production and its relationship to glycolytic rate 3-6. Extracellular acid produced by cells is derived from both lactate, produced by anaerobic glycolysis, and CO2, produced in the citric acid cycle during respiration. For glycolysis, the conversion of glucose to lactate- + H+ and the export of products into the assay medium is the source of glycolytic acidification. For respiration, the export of CO2, hydration to H2CO3 and dissociation to HCO3- + H+ is the source of respiratory acidification. The proportions of glycolytic and respiratory acidification depend on the experimental conditions, including cell type and substrate(s) provided, and can range from nearly 100% glycolytic acidification to nearly 100% respiratory acidification 6. Here, we demonstrate the data collection and calculation methods needed to determine respiratory and glycolytic contributions to total extracellular acidification by whole cells in culture using C2C12 myoblast cells as a model.

Glycolysis is a defining metabolic marker in multiple biological systems. Monitoring glycolysis by measuring the extracellular flux of H+ is common, but requires correction to be quantitative and unambiguous. Here, we demonstrate how to gather and correct extracellular flux data to distinguish between respiratory and glycolytic sources of extracellular acidification.

세포 외 산 생산의 측정 및 분석은 당분 속도를 확인하는

Extracellular measurement of oxygen consumption and acid production is a simple and powerful way to monitor rates of respiration and glycolysis1. Both ...

Novel Production Protocol for Small-scale Manufacture of Probiotic Fermented Foods

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter can be used for the production of fermented milk and other fermented foods either at home or at small-scale in rural settings. For the fresh starter, 1 L of milk is pasteurized in a pan that fits into a larger pan containing water, placed on a source of heat. In this water bath, the milk is heated and incubated at 85 &#176;C for 30 min. Thereafter, the milk is cooled down to 45 &#176;C, transferred to a vacuum flask, inoculated with the dried bacteria and left for at least 16 hr between 30 &#176;C and 45 &#176;C. For the purpose of frequent home production, the fresh starter is frozen into ice cubes, which can be used for the production of small volumes of up to 2 L of fermented milk. For the purpose of small-scale production in resource-poor countries, pasteurization of up to 100 L of milk is conducted in milk cans that are placed in a large sauce pan filled with water and heated on a fire at 85 &#176;C for 30 min, and subsequently cooled to 45 &#176;C. Next, the 100 L batch is inoculated with the 1 L freshly prepared starter mentioned before. To assure an effective fermentation at a temperature between 30 and 45 &#176;C, the milk can is covered with a blanket for 12 hr. For the production of non-dairy fermented foods, the fresh starter is left in a cheese cloth for 12 hr, and the drained-off whey can be subsequently used for the inoculation of a wide range of food raw materials, including vegetables and cereal-based foods.

A production protocol is described for the small-scale production of probiotic fermented dairy drink with the aid of a novel bacterial starter culture.

생균제 발효 식품의 소규모 제조를위한 신규 생산 프로토콜

A novel dried bacterial consortium of Lactobacillus rhamnosus yoba 2012 and Streptococcus thermophilus C106 is cultured in 1 L of milk. This fresh starter ...

A Multi-hole Cryovial Eliminates Freezing Artifacts when Muscle Tissues are Directly Immersed in Liquid Nitrogen

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible cytoplasmic compartments. Another hurdle is the tight apposition of myofibers to the surrounding tissues. Because the process of tissue fixation and paraffin embedding leads to the shrinkage of muscle fibers, freezing is an optimal means of hardening muscle tissue for sectioning. However, a commonly encountered issue, the formation of ice crystals, occurs during the preparation of frozen sections because of the high water content of muscle. The protocol presented here first describes a simple and efficient method for properly freezing muscle tissues by immersing them in liquid nitrogen. The problem with using liquid nitrogen alone is that it causes the formation of a nitrogen gas barrier next to the tissue, which acts as an insulator and inhibits the cooling of the tissues. To avoid this "vapor blanket" effect, a new cryovial was designed to increase the speed of liquid flow around the tissue surface. This was achieved by punching a total of 14 inlet holes in the wall of the vial. According to bubble dynamics, a higher rate of liquid flow results in smaller bubbles and fewer chances to form a gas barrier. When liquid nitrogen flows into the cryovial through the inlet holes, the flow velocity around the tissue is fast enough to eliminate the gas barrier. Compared to the method of freezing muscle tissues using pre-chilled isopentane, this protocol is simpler and more efficient and can be used to freeze muscle in a throughput manner. Furthermore, this method is optimal for institutions that do not have access to isopentane, which is extremely flammable at room temperature.

This protocol describes a procedure to freeze muscle tissues by plunging them directly into liquid nitrogen. This protocol also highlights a new cryovial that can avoid the &#34;blanket effect&#34; of nitrogen gas when liquid nitrogen contacts the tissue surface of a specimen.

근육 조직이 직접 액체 질소에 몰입 할 때 이슈 결빙 멀티 홀 Cryovial 제거

Studies on skeletal muscle physiology face the technical challenge of appropriately processing the specimens to obtain sections with clearly visible ...