여기에 표시된 기술은 에너지학과 (Bioenergy 과학)에서 보조금과 국립 과학 재단 (National Science Foundation)에 의해 자금과 개발되었습니다.

1 부 : 재배와 적합한 식물 자료를 저장 <ol><li> 우리의 경험에서 etiolated 오이 모종의 젊은 hypocotyls이 실험을 위해 세포 벽 재료의 편리한 소스로 제공하고 있습니다. 오이 씨앗 26에서 설정한 일정한 온도 룸에서 어두운 캐비닛에 보관되는 빛을 증거 상자 ° C.에 서부 유럽 표준시 종이에 sown 아르 C가 괜찮을 20 °와 30 ° 사이에 아무것도로 정확한 온도가 중요한 것은 아니지만 온도가 묘목 개발의 적절한 단계에 도달 얼마나 빨리 결정됩니다. 따뜻한 온도, 빨리 묘목이 개발합니다. 그들은 파종 후 3-4일에 도달 길이 약 5cm로 성장 할 때 우리는 일반적으로 모종을 사용합니다. 빛의 심지어 소량 우리가이 기술을 측정되는 묘목 개발의 속도와 세포 벽 특성 모두에 영향을 미칠 때, 묘목이 어둠 속에서 성장하는 것이 중요합니다. 일 3 당신은 세들링 개발에 확인, 희미한 녹색 빛 필터링을 사용하여 상자에 살짝 수 있습니다. </li><li> 모종 신속하게 절단하고 작은 플라스틱 상자, 상자 당 100-150 모종의 포장하고, -80 ° C.에 저장됩니다 이 온도에서 그들은 몇 주 동안 유용하게 남아있다. </li></ol> 2 부 : 준비 셀 벽 샘플 <ol><li> 냉동 컷 모종의 소그룹 (80-10)은 냉동고에서 -80 냉동실 블록을 포함하는 절연 컨테이너로 전송됩니다. </li><li> hypocotyl를 덮고 표피는 카보런덤와 abraded 있습니다. 이것은 반복 서부 유럽 표준시 카보런덤 분말의 굵은 슬러리와 코팅의 엄지와 집게손가락 사이 hypocotyl를 그림으로써 이루어집니다. ; 너무 작은 압력과 표피가 permeabilized되지 않습니다 너무 많은 압력과 표피 레이어가났습니다하고 찢어진 것으로 시작 : 그것은 사용 압력의 정확한 금액을 알고 조금 경험이 걸립니다. 하나는 또한 냉동 hypocotyl 녹아서, 그것이 관리 이완된 및 하드되어 있기 때문에 신속하게 작업합니다. </li><li> 나머지 hypocotyls가 유사한 방식으로 준비하는 동안 abraded hypocotyl는 얼음물에 저장된 후 준수 카보런덤와의 대부분을 제거하는 얼음물에 담근이다. </li><li> hypocotyls는 새로운 하나의 양날 면도날로, 원하는 길이, 보통 1.2 cm로 절단하고 유리 슬라이드에 정렬됩니다. </li><li> 이제 우리는 세포 수액을 제거하고 클램핑 촉진하기 위해 벽면을 평평하게해야합니다. 두 번째 유리 슬라이드는 샌드위치를​​ 형성 8-10 샘플 그룹의 상단에 위치합니다. 무게 (400~500그램)는 5 분 유리 슬라이드 위에 위치합니다. 무게 위해 우리는 정기적으로 물을 적절한 금액을 포함하는 비커를 사용합니다. </li><li> 선택 단계 : 실험에 따라 hypocotyls이 시점에서 간단한 열처리로 inactivated 수 있습니다. 이렇게하려면 우리는 유리가 고무 밴드의 한 쌍을 함께 슬라이드를 바인드 실온에서 드 이온화 물 100 ML있는 용기에 어셈블리를 넣고 전체 전력에서 전자렌지에 넣습니다. 우리 전자렌지로 물이 약 50 S에서 끓기 시작하고 끓는이 시작 후 우리는 전자 레인지 15 S를 중지합니다. 온수는 빠르게 해제 붓고 및 변성을 막을 찬물로 대체됩니다. 귀하의 전자 레인지가 우리와 다를 수 있습니다대로,이 타이밍을 변경할 수 있습니다. 과도한 난방과 샘플이 약하되어 쉽게 휴식. 충분한 가열과 내생 expansin는 inactivated과 벽면 샘플 산성 pH의 몇 가지 대응을 유지하지 않습니다. </li></ol> 파트 3 : 신장계 설정 <ol><li> 벽 샘플은 현재 지속적인 강제 신장계에 고정되어 있습니다. 이것은 벽에 샘플과 샘플의 꼭대기의 끝에 붙어 움직일 수있는 클램프의 기초 끝을 잡고위한 플렉시 쿠베트 구성된 맞춤식 장치입니다. 움직일 수있는 클램프가 위치 센서의 오픈 코일 통과 막대의 끝 부분에 탑재, LVDT 또는 전자 작은 금속 실린더의 위치를​​ 감지 &#39;선형 가변 차동 변압기&#39;, 또는 &#39;핵심&#39;입니다 막대에 연결된. 막대의 위쪽 끝을 조절 평형추있는 레버에 연결되어 있습니다. 이 레버는 벽에 샘플에서 위쪽으로 힘을 조절 금액을 미치는. 힘이 레버의 맨 끝에 조정 금속 무게를 추가하거나 제거하여 조정됩니다. </li><li> 위로 벽면 예제 - hypocotyl 샘플의 기초 끝을 잘 포셉과 혀끝의 끝에 ~ 2~3mm이 움직일 수있는 클램프의 오픈 문턱 사이에 위치로 체포됩니다. 이 클램프 누구의 금속 문턱이 금속 표면과 버퍼 솔루션이나 벽에 샘플 사이의 직접 접촉을 방지하기 위해 플라스틱과 코팅하는 용수철 악어 클램프입니다. 우리의 경험 금속 이온에 연장, 그래서 우리는 금속이 적용 유지 벽의 능력을 발휘 클램프의 리치가 수있다. </li><li> 한 손으로 움직일 수있는 클램프 어셈블리를 개최, 벽 시편의 기초 끝에 이제 플렉시 쿠베트과 쿠베트의 두 기복 조각 사이 였는데입니다조각이 함께 가져온시키고 쿠베트의 벽 샘플의 바닥 끝을 잠금, 꽉 망했다. </li><li> 움직일 수있는 클램프 어셈블리 이제 부드럽게 평형의 전체 힘이 벽에 표본로 전송 있도록 배포되고 있습니다. 우리는 일상적으로 오이 hypocotyls에서 준비 벽 샘플 20g 총 평형추을 사용합니다. 기타 벽 자료 또는 실험 서로 다른 가중치를 필요로 할 수도 있습니다. </li><li> 쿠베트는 버퍼가 가득 (200 UL)과 쿠베트의 위치는 움직일 수있는 클램프가 LVDT 측정 범위의 하단에 회부되도록, 조정 나사와 함께 아래로 이동하거나합니다. 우리 LVDT는 마이크로의 데이터 수집 장치에 연결하고, 그래서 우리는 컴퓨터를 통해 LVDT 위치를 모니터입니다. 우리는 동시에 8 벽에 샘플을 실행할 수 있습니다 하나의 컴퓨터와 병렬로 연결 팔 LVDT 어셈블리 있습니다. 컴퓨터는 매 30 S. 한번 LVDT 어셈블리의 각각의 위치를​​ 기록 </li></ol> 4 부 : 산성 산도 또는 expansin로 측정 확장 응답 - 대표 결과 <ol><li> [첫 번째 실험] 산성 유발 연장의 측정을 위해, 우리는 기본 벽 샘플 (즉, 열과 inactivated되지 않습니다)과 중성 버퍼로 시작은 쿠베트에 추가됩니다. 벽면 샘플 추가 긴장에 대응, 몇 분 동안 연장하지만, 확장 자연 붕괴 몇 분 후에 낮은 요금합니다. 우리 컴퓨터는 우리가 어느 샘플의 길이의 변화 (즉 실험의 시작 후 움직일 수있는 클램프의 위치의 변화입니다) 모니터 또는 우리가 즉, 확장의 속도를 위치의 시간 유도체를 모니터링할 수 있습니다 . 확장 속도는 시간과 낮은 값을 안정화. </li><li> ~ 20 분 후, 중립 버퍼가 제거됩니다. 우리는 벽에 최소한의 방해 또는 기계 조립과, 빨리 버퍼를 제거하는 진공 펌프에 연결된 대형 게이지 피하 바늘로 만든 얇은 금속 파이프를 사용합니다. 산성 버퍼는 다음 산성 버퍼의 전체 교환을 확보하기 위해 1-2 신속한 교류로 때로는 추가됩니다. 그렇다면 우리는 앉아서 벽의 반응을 봐. 일반적으로 우리는 몇 분 안에 확장의 빠른 속도를 검색할 수 있습니다. 어떤 경우에 측정이 더 이상 기간을 연장할 수 있지만 60 분 후에 우리는 일반적으로, 확장 반응을 평가하기 위해 충분한 정보를 얻을 수있다. </li><li> [두번째 실험] expansin 유발 세포 벽 확장의 측정을 위해, 우리는 열을 inactivated 벽 샘플 산성 버퍼로 시작은 쿠베트에 추가됩니다. 첫 번째 실험과 마찬가지로, 세포 벽 확장의 속도가 점차 때문에 기능 expansin 부족의 낮은 값으로 안정. </li><li> ~ 20 분 후, expansin 단백질은 expansin 솔루션의 10-20 UL과 쿠베트에서 버퍼와 &#39;급상승&#39;에 의해 쿠베트에 추가됩니다. expansin는 빠르게 세포 벽 샘플을 관통하고 몇 분 이내에 우리는 확장 률이 증가했습니다 것을 볼 수 있습니다. 이 확장 응답 시간 이상 다음 수 있습니다. </li></ol><img src="/files/ftp_upload/1263/fig1.png" alt="" width="600" height="469" /> 그림 1 : 산성 유발 또는 expansin 유발 벽을 확장을 평가하기 위해 세포 벽 준비 절차의 개요.

<ol>
	<li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+long-term+extension+of+isolated+cell+walls+from+growing+cucumber+hypocotyls.">Characterization of long-term extension of isolated cell walls from growing cucumber hypocotyls.</a> Planta. 177, 121-130 (1989).</li><li>Rayle, D. L., Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Acid+Growth+Theory+of+auxin-induced+cell+elongation+is+alive+and+well.">The Acid Growth Theory of auxin-induced cell elongation is alive and well.</a> Plant Physiol. 99, 1271-1274 (1992).</li><li>Cleland, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Auxin-induced+growth+of+Avena+coleoptiles+involves+two+mechanisms+with+different+pH+optima.">Auxin-induced growth of Avena coleoptiles involves two mechanisms with different pH optima.</a> Plant Physiol. 99, 1556-1561 (1992).</li><li>Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Loosening+of+plant+cell+walls+by+expansins.">Loosening of plant cell walls by expansins.</a> Nature. 407, 321-326 (2000).</li><li>McQueen-Mason, S., Durachko, D. M., Cosgrove, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Two+endogenous+proteins+that+induce+cell+wall+expansion+in+plants.">Two endogenous proteins that induce cell wall expansion in plants.</a> Plant Cell. 4, 1425-1433 (1992).</li></ol>

그들이 처리하고 좋은 감도와 반응하기 쉬운 벽 샘플의 신뢰할 수있는 원본으로 입증해야하기 때문에이 실험을 위해 우리는 오이 hypocotyls의 세포 벽를 사용했습니다. 우리는 또한 어린 시금치 잎과 셀러리 줄기로 슈퍼마켓에서 다른 모종뿐만 아니라 재료에서 벽 좋은 성공을 있었다. 기본적으로, 젊고 부드럽고 빠르게 성장하는 식물의 조직이 쉽게이 기술을 측정 가능성이 있지만, 거친, 이전, ?...

measuring plant cell wall extension (creep) induced by acidic ph and by alpha-expansin

성장 식물 세포 벽 특질상 우리가 그들이 낮은 PH (&lt;5) 1에서 더 확장 의미하는 &#39;산성의 성장&#39;으로 알려진 속성을 나타냅니다. 식물 호르몬 옥신은 급격히 산성 성장 메커니즘 2, 3 적어도 일부 젊은 줄기 및 유사 조직의 세포 신장을 자극. 옥신은 H를 활성화 + 플라즈마 막에 펌프를, 세포 벽 솔루션의 산성화을 불러 일으킨다. 벽면 산성화은 세포 벽이 셀 turgor 압력에 의해 만들어진 벽면 긴장에 항복로 인해, 내생 세포 벽 - 느슨해진 단백질 4아르 expansins를 활성화됩니다. 그 결과, 세포가 급속하게 확대하기 시작합니다. 이 &#39;산성 성장&#39;현상이 쉽게 격리 (nonliving) 세포 벽 표본 단위로 측정됩니다. 산성 유도 확장자를 받아야하는 세포 벽의 능력은 단순히 세포 벽의 다당류 (예 : pectins)의 구조 배열의 결과 아니라, expansins 5 활동에 따라 달라집니다. Expansins은 알려진 효소 활동을하지 않으며 expansin 활동에 대해 분석하는 유일한 방법은 세포 벽 확장 자신의 유도를 측정하는 것입니다. 이 비디오 보고서는 세부 expansin의 assays에 적합한 벽 자료를 얻기위한 소스와 준비 기술과 산성 유발 확장 및 성장 오이 hypocotyls에서 준비 벽 샘플 expansin 유발 확장을 보여주기에갑니다. 적당한 세포 벽 샘플을 얻으려면, 오이 모종이 어둠 속에서 재배, hypocotyls가 -80에서 잘라 냉동 ° C. 냉동 hypocotyls는 abraded 평평하고 신장계 측정을위한 특별한 쿠베트에 일정한 장력에 고정되어 있습니다. 산성 유도 확장을 측정하려면 벽은 처음에 기본 셀 벽면의 구성 요소 expansins의 낮은 활동의 결과, 중성 산도에서 버퍼입니다. 산성 산도에 버퍼 교환시 expansins가 활성화하고 세포 벽이 급속하게 확장하고 있습니다. 우리는 또한 reconstitution 분석에 expansin 활동을 보여줍니다. 이 부분에 대한, 우리는 세포 벽 샘플에서 네이티브 expansins을 변성하는 간단한 열처리를 사용합니다. 이러한 inactivated 세포 벽 산성 버퍼에도 확장할 수 없지만, 세포 벽에 expansins의 또한 급속하게 확장하는 능력을 복원합니다.

Watch this Scientific Journal Video about Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin at JoVE.com

Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

우리는 산성 버퍼와 expansin 단백질에 의해 유도된 식물 세포 벽의 표본의 장기 연장 (크리프)를 측정하기 위해 일정한 - 강제 신장계의 사용을 보여줍니다.

측정 식물 세포 벽 확장 (크리프)는 산성 산도에 의해 및 알파 Expansin에 의해 유도된

Growing plant cell walls characteristically exhibit a property known as 'acid growth', by which we mean they are more extensible at low pH (&lt; 5) 1. The ...

measuring-plant-cell-wall-extension-creep-induced-acidic-ph-alpha

Research

JoVE Journal

Biology

Measuring Plant Cell Wall Extension (Creep) Induced by Acidic pH and by Alpha-Expansin

13.9K 조회수.  Penn State University. 우리는 산성 버퍼와 expansin 단백질에 의해 유도된 식물 세포 벽의 표본의 장기 연장 (크리프)를 측정하기 위해 일정한 - 강제 신장계의 사용을 보여줍니다.

비디오: Measuring Plant Cell Wall Extension Creep Induced by Acidic pH and by Alpha-Expansin

Title Cell Encapsulation by Droplets

흩뿌려진의 제목 셀 캡슐화

연구

생물학

Virus-induced Gene Silencing (VIGS) in Nicotiana benthamiana and Tomato

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA regulators: microRNAs, which are produced from non-protein coding genes and short interfering RNAs (siRNAs). Plants use RNAi to control transposons and to exert tight control over developmental processes such as flower organ formation and leaf development2,3,4. Plants also use RNAi to defend themselves against infection by viruses. Consequently, many viruses have evolved suppressors of gene silencing to allow their successful colonization of their host5.
Virus-induced gene silencing (VIGS) is a method that takes advantage of the plant RNAi-mediated antiviral defense mechanism. In plants infected with unmodified viruses the mechanism is specifically targeted against the viral genome. However, with virus vectors carrying sequences derived from host genes, the process can be additionally targeted against the corresponding host mRNAs. VIGS has been adapted for high-throughput functional genomics in plants by using the plant pathogen Agrobacterium tumefaciens to deliver, via its Ti plasmid, a recombinant virus carrying the entire or part of the gene sequence targeted for silencing. Systemic virus spread and the endogenous plant RNAi machinery take care of the rest. dsRNAs corresponding to the target gene are produced and then cleaved by the ribonuclease Dicer into siRNAs of 21 to 24 nucleotides in length. These siRNAs ultimately guide the RNA-induced silencing complex (RISC) to degrade the target transcript2.
Different vectors have been employed in VIGS and one of the most frequently used is based on tobacco rattle virus (TRV). TRV is a bipartite virus and, as such, two different A. tumefaciens strains are used for VIGS. One carries pTRV1, which encodes the replication and movement viral functions while the other, pTRV2, harbors the coat protein and the sequence used for VIGS6,7. Inoculation of Nicotiana benthamiana and tomato seedlings with a mixture of both strains results in gene silencing. Silencing of the endogenous phytoene desaturase (PDS) gene, which causes photobleaching, is used as a control for VIGS efficiency. It should be noted, however, that silencing in tomato is usually less efficient than in N. benthamiana. RNA transcript abundance of the gene of interest should always be measured to ensure that the target gene has efficiently been down-regulated. Nevertheless, heterologous gene sequences from N. benthamiana can be used to silence their respective orthologs in tomato and vice versa8.

Virus-induced Gene Silencing VIGS in Nicotiana benthamiana and Tomato

Description of a virus-induced gene silencing (VIGS) method for knock-down of gene expression in Nicotiana benthamiana and tomato.

바이러스 유도 진 입을 (VIGS)에 Nicotiana benthamiana 토마토

RNA interference (RNAi) is a highly specific gene-silencing phenomenon triggered by dsRNA1. This silencing mechanism uses two major classes of RNA ...

Identification of Growth Inhibition Phenotypes Induced by Expression of Bacterial Type III Effectors in Yeast

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within the cell, type III effectors subvert host cellular processes to suppress immune responses and promote pathogen growth. Numerous type III effectors of plant and animal bacterial pathogens have been identified to date, yet only a few of them are well characterized. Understanding the functions of these effectors has been undermined by a combination of functional redundancy in the effector repertoire of a given bacterial strain, the subtle effects that they may exert to increase virulence, roles that are possibly specific to certain infection stages, and difficulties in genetically manipulating certain pathogens. Expression of type III effectors in the budding yeast Saccharomyces cerevisiae may allow circumventing these limitations and aid to the functional characterization of effector proteins. Because type III effectors often target cellular processes that are conserved between yeast and other eukaryotes, their expression in yeast may result in growth inhibition phenotypes that can be exploited to elucidate effector functions and targets. Additional advantages to using yeast for functional studies of bacterial effectors include their genetic tractability, information on predicted functions of the vast majority of their ORFs, and availability of numerous tools and resources for both genome-wide and small-scale experiments. Here we discuss critical factors for designing a yeast system for the expression of bacterial type III effector proteins. These include an appropriate promoter for driving expression of the effector gene(s) of interest, the copy number of the effector gene, the epitope tag used to verify protein expression, and the yeast strain. We present procedures to induce expression of effectors in yeast and to verify their expression by immunoblotting. In addition, we describe a spotting assay on agar plates for the identification of effector-induced growth inhibition phenotypes. The use of this protocol may be extended to the study of pathogenicity factors delivered into the host cell by any pathogen and translocation mechanism.

In this video, we describe a procedure for the expression of bacterial type III effectors in yeast and the identification of effector-induced growth inhibition phenotypes. Such phenotypes can be subsequently exploited to elucidate effector functions and targets.

성장 저해 Phenotypes의 확인은 효모에서 세균의 종류 III Effectors 표현에 의해 유도된

Many Gram-negative pathogenic bacteria use a type III secretion system to translocate a suite of effector proteins into the cytosol of host cells. Within ...

Agar-Block Microcosms for Controlled Plant Tissue Decomposition by Aerobic Fungi

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; agar-block). It is well-accepted that soil-block microcosms yield higher decay rates, fewer moisture issues, lower variability among studies, and higher thresholds of preservative toxicity. Soil-block testing is thus the more utilized technique and has been standardized by American Society for Testing and Materials (ASTM) (method D 1413-07). The soil-block design has drawbacks, however, using locally-variable soil sources and in limiting the control of nutrients external (exogenous) to the decaying tissues. These drawbacks have emerged as a problem in applying this method to other, increasingly popular research aims. These modern aims include degrading lignocellulosics for bioenergy research, testing bioremediation of co-metabolized toxics, evaluating oxidative mechanisms, and tracking translocated elements along hyphal networks. Soil-blocks do not lend enough control in these applications. A refined agar-block approach is necessary.
Here, we use the brown rot wood-degrading fungus Serpula lacrymans to degrade wood in agar-block microcosms, using deep Petri dishes with low-calcium agar. We test the role of exogenous gypsum on decay in a time-series, to demonstrate the utility and expected variability. Blocks from a single board rip (longitudinal cut) are conditioned, weighed, autoclaved, and introduced aseptically atop plastic mesh. Fungal inoculations are at each block face, with exogenous gypsum added at interfaces. Harvests are aseptic until the final destructive harvest. These microcosms are designed to avoid block contact with agar or Petri dish walls. Condensation is minimized during plate pours and during incubation. Finally, inoculum/gypsum/wood spacing is minimized but without allowing contact. These less technical aspects of agar-block design are also the most common causes of failure and the key source of variability among studies. Video publication is therefore useful in this case, and we demonstrate low-variability, high-quality results.

This video demonstrates a controlled environment approach to study degradation of lignocellulosic plant tissues by aerobic fungi. The ability to control nutrient sources and moisture is a key advantage of agar-block microcosms, but the approach often yields mixed success. We address critical pitfalls to yield reproducible, low-variability results.

에어로빅 곰팡이에 의해 제어 플랜트 조직 분해에 대한 한천 - 블럭 Microcosms

The two principal methods for studying fungal biodegradation of lignocellulosic plant tissues were developed for wood preservative testing (soil-block; ...

Pouring and Running a Protein Gel by reusing Commercial Cassettes

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry and molecular biology researchers1-4. For laboratories that perform daily analyses of proteins, the cost of commercially available polyacrylamide gels (&tilde;$10/gel) can be considerable over time. To mitigate this cost, some researchers prepare their own polyacrylamide gels. Traditional methods of pouring these gels typically utilize specialized equipment and glass gel plates that can be expensive and preclude pouring many gels and storing them for future use. Furthermore, handling of glass plates during cleaning or gel pouring can result in accidental breakage creating a safety hazard, which may preclude their use in undergraduate laboratory classes. Our protocol demonstrates how to pour multiple protein gels simultaneously by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment. In addition, plastic gel cassettes are extremely resistant to breakage, which makes them ideal for undergraduate laboratory classrooms.

Our protocol demonstrates how to pour multiple protein gels at a time by recycling Invitrogen Nupage Novex minigel cassettes, and inexpensive materials purchased at a home improvement store. This economical and streamlined method includes a way to store the gels at 4&deg;C for a few weeks. By re-using the plastic gel cassettes from commercially available gels, labs that run frequent protein gels can save significant costs and help the environment.

상업 카세트를 재사용하여 물을 붓는과 단백질 젤 러닝

The evaluation of proteins using sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis is a common technique used by biochemistry ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Microarrays를 사용하여 식물 세포의 벽 폴리머의 Glycan 프로파일

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) maintain the turnover of all mature blood cell lineages. The coordination of the complex signals leading to specific HSC fates relies upon the interaction between HSCs and the intricate bone marrow microenvironment, which is still poorly understood[1-2].
We describe how by combining a newly developed specimen holder for stable animal positioning with multi-step confocal and two-photon in vivo imaging techniques, it is possible to obtain high-resolution 3D stacks containing HSPCs and their surrounding niches and to monitor them over time through multi-point time-lapse imaging. High definition imaging allows detecting ex vivo labeled hematopoietic stem and progenitor cells (HSPCs) residing within the bone marrow. Moreover, multi-point time-lapse 3D imaging, obtained with faster acquisition settings, provides accurate information about HSPC movement and the reciprocal interactions between HSPCs and stroma cells.
Tracking of HSPCs in relation to GFP positive osteoblastic cells is shown as an exemplary application of this method. This technique can be utilized to track any appropriately labeled hematopoietic or stromal cell of interest within the mouse calvarium bone marrow space.

In Vivo 4-Dimensional Tracking of Hematopoietic Stem and Progenitor Cells in Adult Mouse Calvarial Bone Marrow

The nature of the interactions between hematopoietic stem and progenitor cells (HSPCs) and bone marrow niches is poorly understood. Custom hardware modifications and a multi-step acquisition protocol allow the use of two-photon and confocal microscopy to image ex vivo labeled HSPCs homed within bone marrow areas, tracking interactions and movement.

성인 마우스 두개골 골수에서 조혈 줄기 및 전구 세포의 생체 내 4 차원 추적에서

Through a delicate balance between quiescence and proliferation, self renewal and production of differentiated progeny, hematopoietic stem cells (HSCs) ...

Fecal Glucocorticoid Analysis: Non-invasive Adrenal Monitoring in Equids

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, corticotrophin releasing hormone (CRH) is released from the hypothalamus in the brain. This stimulates the release of adrenocorticotrophic hormone (ACTH) from the pituitary gland, which in turn stimulates release of glucocorticoids from the adrenal gland. In horses the glucocorticoid corticosterone is responsible for several adaptations needed to support equine flight behaviour and subsequent removal from the aversive situation. Corticosterone metabolites can be detected in the feces of horses and assessment offers a non-invasive option to evaluate long term patterns of adrenal activity. Fecal assessment offers advantages over other techniques that monitor adrenal activity including blood plasma and saliva analysis. The non-invasive nature of the method avoids sampling stress which can confound results. It also allows the opportunity for repeated sampling over time and is ideal for studies in free ranging horses. This protocol describes the enzyme linked immunoassay (EIA) used to assess feces for corticosterone, in addition to the associated biochemical validation.

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. The method offers a non-invasive option to assess long term patterns in both domestic and free ranging horses. This protocol describes the enzyme linked immunoassay involved and the associated biochemical validation.

대변​​ 글루코 코르티코이드 분석 : equids는 비 침습적 부신 모니터링

Adrenal activity can be assessed in the equine species by analysis of feces for corticosterone metabolites. During a potentially aversive situation, ...

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix containing polysaccharides that include cellulose microfibrils that form both crystalline structures and cellulose chains of amorphous organization. The orientation of the cellulose fibers and their concentrations dictate the mechanical properties of the cell. Several methods are used to determine the levels of crystalline cellulose, each bringing both advantages and limitations. Some can distinguish the proportion of crystalline regions within the total cellulose. However, they are limited to whole-organ analyses that are deficient in spatiotemporal information. Others relying on live imaging, are limited by the use of imprecise dyes. Here, we report a sensitive polarized light-based system for specific quantification of relative light retardance, representing crystalline cellulose accumulation in cross sections of Arabidopsis thaliana roots. In this method, the cellular resolution and anatomical data are maintained, enabling direct comparisons between the different tissues composing the growing root. This approach opens a new analytical dimension, shedding light on the link between cell wall composition, cellular behavior and whole-organ growth.

High Resolution Quantification of Crystalline Cellulose Accumulation in Arabidopsis Roots to Monitor Tissue-specific Cell Wall Modifications

Crystalline cellulose is an important constituent of the plant cell wall. However, its quantification at a cellular resolution is technically challenging. Here, we report the use of polarized light technology and root cross sections to obtain information of cell wall composition at a spatiotemporal resolution.

에서 결정 셀룰로오스 축적의 높은 해상도 정량<em&gt; 애기 장대</em&gt; 뿌리 조직 특이 세포 벽 수정을 모니터링하는

Plant cells are surrounded by a cell wall, the composition of which determines their final size and shape. The cell wall is composed of a complex matrix ...

High-throughput CRISPR Vector Construction and Characterization of DNA Modifications by Generation of Tomato Hairy Roots

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for functional genomics studies. While most cloning strategies are simple to perform, they generally use multiple steps and can require several days to generate the ultimate constructs. The method presented here is based on DNA assembly and can produce fully functional CRISPR vectors in a single cloning reaction. Vector construction can also be pooled, further increasing the efficiency and utility of the process. A modification of the method is used to create CRISPR vectors with multiple gene targets. CRISPR vectors are then transformed into tomato hairy roots to generate transgenic materials with targeted DNA modifications. Hairy roots are a useful system for testing vector functionality as they are technically simple to generate and amenable to large-scale production. The methods presented here will have wide application as they can be used to generate a variety of CRISPR vectors and be used in a wide range of plant species.

Using DNA assembly, multiple CRISPR vectors can be constructed in parallel in a single cloning reaction, making the construction of large numbers of CRISPR vectors a simple task. Tomato hairy roots are an excellent model system to validate CRISPR vectors and generate mutant materials.

토마토 털이 뿌리의 생성에 의한 높은 처리량 CRISPR 벡터 건설 및 DNA 수정의 특성

Targeted DNA mutations generated by vectors with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technology have proven useful for ...