본 논문은 여기에서 설명한 원래 연구를 호스팅하는 후반 박사 Zvi 셀린저의 기억에 전념하고 있습니다.

브래드 포드 단백질 보정 그래프의 선형화 : 일반적으로 브래드 포드 분석 1로 알려진 쿠매시 화려한 파란색 단백질 분석은, 널리 때문에 신속하고 편리한 프로토콜뿐만 아니라 그 상대 감도의하는 데 사용됩니다. 불행히도, 단백질 농도의 광범위한 (그림 1) 이상의 곡률의 큰 정도가 있습니다. 따라서, 상대적으로 높은 단백질 농도, 20-10 MG / ML BSA의 단지 좁은 범위는 다음 더 나은 (그림 1, 녹색) 선형 회귀에 맞는 보정 그래프에 대한 사용됩니다. 그러나, 비선 형성은 큰 오류를 방지하기 위해 교정 그래프의 제한된 범위 내에서 가을에 알려지지 않은 샘플의 단백질 농도를 필요로하고 또한 제한된 범위의 정확성을 줄일 수 있습니다. 비선 형성은 단백질의 마이크로 그램 금액은 사용할 수 없습니다 특히 심각한 문제를 제시하고, 그것은 종종 알려지지 않은 샘플의 여러 dilutions이 필요합니다. 마찬가지로 원래 브래드 포드 종이에 명시된 바와 같이, &quot;염료의 두 가지 색상 형태의 스펙트럼에 중복이되기 때문에 비선 형성의 원인은 시약 자체입니다.&quot;1. 사실, 쿠매시 화려한 파란색 색소의 세 형태 분석 2 일반적인 산성 산도에서 산성 - 기본 평형에 있습니다. 빨간색, 파란색, 그리고 녹색 양식, 470에서 각각 590과 650 nm의 (그림 2) 흡광도 맥시멈 있습니다. 파란 달아 NM 3, 4 (그림 2) 594에서 빛을 흡수 복잡한을 형성 단백질 바인딩 형식입니다. 브래드 포드는 또한 1 (그림 3) &quot;더 많은 염료는 단백질 바인딩으로 시약의 배경 값이 지속적으로 감소하고있다&quot;고 지적했다. 증가 단백질 수량이 추가로 따라서, 우리는 단백질 염색 단지가 흡수하지 않는 450 nm의에서 흡광도의 변화를 측정하여 590 nm의 배경의 감소를 계산하기 위해 시도했습니다. 우리는 발견 감소 배경 부분 것이 아니지만 완전히, 비선 형성 (그림 4)를 차지하고 있습니다. 단백질 염색 바인딩은 평형 5와 같이 있기 때문에 우리는 그때, 무료 염료 농도의 감소가 선형 응답의 또 다른 왜곡을 생성하는 가상, 복잡한 형성은뿐만 아니라 무료로 염료의에서뿐만 아니라 무료로 단백질의 농도에 따라 달라집니다 (그림 5). 계정에 무료 염료의 변수 농도와 관련된 두 문제를 복용, 우리는 단백질 농도와 흡광도 측정, 450 NM (그림 6)을 통해 590 nm의 비율 사이의 선형 관계를 설명하는 수학 방정식을 개발했습니다. 이론 및 실험적 연구에 대한 자세한 설명은 분석 생화학 6 우리 1,996 간행물에서 찾을 수 있습니다. 수학 방정식은 실험적으로 테스트하여 전체 단백질 농도 범위 (그림 7)을 통해 선형 보정 곡선을 얻을 것으로 판명되었다. 또한 방정식은 Y 축 절편 6 올바른 산도에 의존 가치의 독립적인 결정에 의해도 확인되었다. 향상된 브래드 포드 단백질 분석에 대한 자세한 프로토콜, Microplate의 흡광도 Reader를 사용하여 <ol><li> 표준 소 혈청 알부민의 0.1 MG / ML 재고 솔루션을 준비합니다. 다른 표준은 선택하지만, 같은 표준은 모든 실험에서 사용해야합니다 수 있습니다. </li><li> 탈이온수에있는 미지의 샘플을 희석. 5-50 μg / ML하는 것을 목표로하고 있습니다. 분석의 선형 범위에 대한 명백한 제한이 없기 때문에 아직 높거나 낮은 단백질 농도가 허용됩니다. 그러나, 측정 흡광도 리더의 선형 범위 내에서해야합니다. </li><li> 탈이온수의 브래드 포드 시약 (바이오 래드)가 2.5 배 희석. </li><li> 0 세중의의 우물 (0-5 μg BSA 보정 곡선을 만드는)에 BSA 주식 솔루션의 10-50 μl를 추가합니다. / 잘 100 μl에 도달 탈이온수로 보완합니다. </li><li> 다른 우물에서 triplicates에 알려지지 않은 샘플 100 μl를 추가합니다. 미지 시료의 농도는 몇 가지 정확성을 높이기 위해 사용할 수 있습니다. </li><li> 모든 우물에 희석 브래드 포드 시약 100 μl를 추가합니다. 총 볼륨 잘 200 μl / 수 있습니다. </li><li> 적어도 5 분 아니지만 색상 개발을위한 이상 60 분 정도 기다립니다. </li><li> 빈은 탈이온수 (가 아니라 잘 제로 단백질 색소) 200 μl해야합니다. </li><li> 590 NM에서 450 nm의 흡광도에서를 측정. </li><li> 590 nm의 흡광도에서 네트워크의 가치를 나누어 450 nm의에서 보정 그래프를 준비합니다. 제로 단백질 (단 염료) 값이 데이터 포인트 (그림 8)로 포함되어야합니다. </li><li> 교정 곡선 (그림 9)의 선형 방정식을 바탕으로 미지 시료의 농도를 계산합니다. </li></ol> 대표 결과 : 590 NM의 단일 파장, 흡광도 값을 450 nm의 이상의 590 nm의 비율에서 흡광도와는 달리, 선형 w는ith 단백질 농도 (그림 10). 미지의 시료의 단백질 농도는 단순히 교정 곡선 (그림 10, 방정식)의 선형 방정식을 사용하여 계산됩니다. 그러나, 증가 정확도는 몇 가지 알려지지 않은 샘플 dilutions를 측정하여 얻을 수 있습니다. 이를 위해 두 개의 그래프를 준비합니다. 첫 번째는 X 축 (그림 11, 오른쪽)에서 μg 단백질과 표준 교정 그래프입니다. 두 번째 그래프는 X 축 (그림 11, 왼쪽)에있는 원래 undiluted 샘플 μl와 함께 미지의 샘플입니다. 염료 전용 값은 (그림 11) 두 그래프에 포함되어야합니다. 미지의 시료의 단백질 농도는 미지의 시료와 표준의 슬로프 (그림 11, 방정식)을 나누어 파생됩니다. <img src="/files/ftp_upload/1918/1918fig2.jpg" alt="그림 1" /> 그림 1. 기존 브래드 포드 교정 그래프. 선형 범위는 녹색 기호로 표시됩니다. <img src="/files/ftp_upload/1918/1918fig1.jpg" alt="그림 2" /> 그림 2. 단백질 염색 복합 혼자 염료의 스펙트럼. <img src="/files/ftp_upload/1918/1918fig3.jpg" alt="그림 3" /> 그림 3. 단백질 염색 평형. <img src="/files/ftp_upload/1918/1918fig4.jpg" alt="그림 4" /> 그림 4. 정확한 계산 배경 감소는 부분 비선 형성을 감소시킵니다. <img src="/files/ftp_upload/1918/1918fig5.jpg" alt="그림 5" /> 그림 5. 단백질 염색 평형. <img src="/files/ftp_upload/1918/1918fig6.jpg" alt="그림 6" /> 그림 6. 브래드 포드 단백질 분석의 선형화을위한 수학 기초. <img src="/files/ftp_upload/1918/1918fig7.jpg" alt="그림 7" /> 그림 7. 브래드 포드 보정 그래프의 선형화. <img src="/files/ftp_upload/1918/1918fig8.jpg" alt="그림 8" /> 그림 8. 흡광도 비율 값 계산. <img src="/files/ftp_upload/1918/1918fig9.jpg" alt="그림 9" /> 그림 9. 리니어 브래드 포드 교정 그래프. <img src="/files/ftp_upload/1918/1918fig10.jpg" alt="그림 10" /> 그림 10. 브래드 포드 보정 그래프의 선형화. <img src="/files/ftp_upload/1918/1918fig11.jpg" alt="그림 11" /> 그림 11. 불명 샘플 농도 계산.

<ol>
	<li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal Biochem. 72, 248-254 (1976).</li><li>Chial, H. J., Thompson, H. B., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+spectral+study+of+the+charge+forms+of+Coomassie+blue+G.">A spectral study of the charge forms of Coomassie blue G.</a> Anal Biochem. 209, 258-266 (1993).</li><li>Chial, H. J., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+the+binding+of+Coomassie+brilliant+blue+to+proteins+at+low+and+neutral+pH.">A comparison of the binding of Coomassie brilliant blue to proteins at low and neutral pH.</a> Anal Biochem. 213, 362-369 (1993).</li><li>Compton, S. J., Jones, C. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanism+of+dye+response+and+interference+in+the+Bradford+protein+assay.">Mechanism of dye response and interference in the Bradford protein assay.</a> Anal Biochem. 151, 369-374 (1985).</li><li>Congdon, R. W., Muth, G. W., Splittgerber, A. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+binding+interaction+of+Coomassie+blue+with+proteins.">The binding interaction of Coomassie blue with proteins.</a> Anal Biochem. 213, 407-413 (1993).</li><li>Zor, T., Selinger, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Linearization+of+the+Bradford+protein+assay+increases+its+sensitivity:+theoretical+and+experimental+studies.">Linearization of the Bradford protein assay increases its sensitivity: theoretical and experimental studies.</a> Anal Biochem. 236, 302-308 (1996).</li></ol>

브래드 포드 단백질 분석 인해 성능과 상대 감도 자사의 용이성으로 인기가 있습니다. 프로토콜에 의해 얻은 범위 전체 단백질 농도 이상의 선형화는 미지 샘플의 교정 그래프의 범위 내에서 빠지지하지 않아도 여기에 추가로, 분석을 간단하게 제시. 중요한 것은 향상된 프로토콜은 더 이상 원래 브래드 프로토콜에 비해 다음과 같은 장점을 제공합니다 : <ol><li> 정확도를 ...

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<td>Bradford reagent</td>
<td>&nbsp;</td>
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<td>Bovine Serum Albumin</td>
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linearization of the bradford protein assay

브래드 쿠매시 화려한 파란색 분석에서 단백질의 마이크로 그램 수량의 결정은 590 nm의 흡광도에서 측정하여 수행됩니다. 이것은 가장 일반적인 분석은 생화 학적 측정의 정상화를 목적으로, 세포 lysates, 휴대 분수, 또는 재조합 단백질 샘플에 신속하고 간단하게 단백질 부량 수 있습니다. 그러나, 본질적 비선 형성이 방법의 감도와 정확성을 타협. 이것은 표준 분석 조건 하에서 흡광도 590 NM에서 측정과 450 nm의 비율은 단백질 농도와 엄격하게 선형임을 표시됩니다. 이 간단한 절차는 정확성을 증가 및 아래쪽 소 혈청 알부민 50 NG 10 배, 좋으 부량에 대한 분석의 감도를 향상시킵니다. 또한, 일반적으로 세포 lysates을 만드는 데 사용되는 세제의 도입 간섭은 크게 새로운 프로토콜에 의해 줄어 듭니다. 대량 작업 및 맥주의 율법에 기초하여 개발된 선형 방정식 완벽하게 실험 데이터를 맞는 데요.

Watch this Scientific Journal Video about Linearization of the Bradford Protein Assay at JoVE.com

Linearization of the Bradford Protein Assay

신속하고 편리한 브래드 포드 분석에 의해 단백질 결정의 정확성과 감도는 본질적인 비선 형성에 의해 손상됩니다. 우리는 크게, 정확성을 증가 10 배에 대한 분석의 감도를 향상하고, 크게 세제에 의해 간섭을 줄일 수있는 간단한 선형화 절차를 보여줍니다.

브래드 포드 단백질 분석의 선형화

Determination of microgram quantities of protein in the Bradford Coomassie brilliant blue assay is accomplished by measurement of absorbance at 590 nm. ...

linearization-of-the-bradford-protein-assay

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JoVE Journal

Biology

97.9K 조회수.  Tel Aviv University. 신속하고 편리한 브래드 포드 분석에 의해 단백질 결정의 정확성과 감도는 본질적인 비선 형성에 의해 손상됩니다. 우리는 크게, 정확성을 증가 10 배에 대한 분석의 감도를 향상하고, 크게 세제에 의해 간섭을 줄일 수있는 간단한 선형화 절차를 보여줍니다.

비디오: Linearization of the Bradford Protein Assay

Actin Co-Sedimentation Assay; for the Analysis of Protein Binding to F-Actin

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates tension and helps maintains cellular shape.  Although the actin cytoskeleton is a rigid structure, it is a dynamic structure that is constantly remodeling.  A number of proteins can bind to the actin cytoskeleton.  The binding of a particular protein to F-actin is often desired to support cell biological observations or to further understand dynamic processes due to remodeling of the actin cytoskeleton. The actin co-sedimentation assay is an in vitro assay routinely used to analyze the binding of specific proteins or protein domains with F-actin.  The basic principles of the assay involve an incubation of the protein of interest (full length or domain of) with F-actin, ultracentrifugation step to pellet F-actin and analysis of the protein co-sedimenting with F-actin.  Actin co-sedimentation assays can be designed accordingly to measure actin binding affinities and in competition assays.

Proteins bind to filamentous actin (F-actin) through distinct actin binding modules. In this video we demonstrate the procedure of actin co-sedimentation, which is an in vitro assay routinely used to analyze proteins or specific domains that bind F-actin.

굴지 공동 침강 분석, F - 굴지에 바인딩 단백질의 분석

The actin cytoskeleton within the cell is a network of actin filaments that allows the movement of cells and cellular processes, and that generates ...

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생물학

Detection of Protein Ubiquitination

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of three enzymes. They include ubiquitin-activating enzyme E1, ubiquitin-conjugating enzyme E2, and ubiquitin ligase E3. Unlike to E1 and E2, E3 ubiquitin ligases display substrate specificity. On the other hand, numerous deubiquitylating enzymes have roles in processing polyubiquitinated proteins. Ubiquitination can result in change of protein stability, cellular localization, and biological activity. Mutations of genes involved in the ubiquitination/deubiquitination pathway or altered ubiquitin system function are associated with many different human diseases such as various types of cancer, neurodegeneration, and metabolic disorders. The detection of altered or normal ubiquitination of target proteins may provide a better understanding on the pathogenesis of these diseases. &nbsp;Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro. These protocols are also useful to detect other ubiquitin-like small molecule modification such as sumolyation and neddylation.

Ubiquitination is a key posttranslational modification carried out by a set of three enzymes. Mutations of genes involved in this modification are associated with many different human diseases. Here, we describe protocols to detect protein ubiquitination in cultured cells in vivo and test tubes in vitro.

단백질 Ubiquitination의 감지

Ubiquitination, the covalent attachment of the polypeptide ubiquitin to target proteins, is a key posttranslational modification carried out by a set of ...

Staining of Proteins in Gels with Coomassie G-250 without Organic Solvent and Acetic Acid

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents (Methanol, Ethanol or 2-Propanol) and acetic acid are used for fixation, staining and destaining of proteins in a gel after SDS-PAGE. To speed up the procedure, heating the staining solution in the microwave oven for a short time is frequently used. This usually results in evaporation of toxic or hazardous Methanol, Ethanol or 2-Propanol and a strong smell of acetic acid in the lab which should be avoided due to safety considerations. In a protocol originally published in two patent applications by E.M. Wondrak (US2001046709 (A1), US6319720 (B1)), an alternative composition of the staining solution is described in which no organic solvent or acid is used. The CBB is dissolved in bidistilled water (60-80mg of CBB G-250 per liter) and 35 mM HCl is added as the only other compound in the staining solution. The CBB staning of the gel is done after SDS-PAGE and thorough washing of the gel in bidistilled water. By heating the gel during the washing and staining steps, the process can be finished faster and no toxic or hazardous compunds are evaporating. The staining of proteins occurs already within 1 minute after heating the gel in staining solution and is fully developed after 15-30 min with a slightly blue background that is destained completely by prolonged washing of the stained gel in bidistilled water, without affecting the stained protein bands.

A short protocol for protein staining with Coomassie Brilliant Blue (CBB) G-250 in polyacrylamide gels is described without using organic solvents or acetic acid as in the classical staining procedures with CBB.

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

In classical protein staining protocols using Coomassie Brilliant Blue (CBB), solutions with high contents of toxic and flammable organic solvents ...

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

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

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

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 ...

Bimolecular Fluorescence Complementation (BiFC) Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular environment. The Bimolecular Fluorescence Complementation (BiFC) Assay1 allows researchers to visualize protein-protein interactions in living cells and has become an essential research tool. This assay is based on the facilitated association of two fragments of a fluorescent protein (GFP) that are each fused to a potential interacting protein partner. The interaction of the two protein partners would facilitate the association of the N-terminal and C-terminal fragment of GFP, leading to fluorescence. For plant researchers, onion epidermal cells are an ideal experimental system for conducting the BiFC assay because of the ease in obtaining and preparing onion tissues and the direct visualization of fluorescence with minimal background fluorescence. The Helios Gene Gun (BioRad) is commonly used for bombarding plasmid DNA into onion cells. We demonstrate the use of Helios Gene Gun to introduce plasmid constructs for two interacting Arabidopsis thaliana transcription factors, SEUSS (SEU) and LEUNIG HOMOLOG (LUH)2 and the visualization of their interactions mediated by BiFC in onion epidermal cells.

Bimolecular Fluorescence Complementation BiFC Assay for Protein-Protein Interaction in Onion Cells Using the Helios Gene Gun

This article illustrates how to properly use the BioRad Helios Gene Gun to introduce plasmid DNA into onion epidermal cells and how to test for protein-protein interactions in onion cells based on the principle of Bimolecular Fluorescence Complementation (BiFC)

헬리 우스 진 건을 사용하여 양파 세포에서 단백질 - 단백질 상호 작용에 대한 Bimolecular 형광 Complementation (BiFC) 어세이

Investigation of gene function in diverse organisms relies on knowledge of how the gene products interact with each other in their normal cellular ...

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 ...

Linking Predation Risk, Herbivore Physiological Stress and Microbial Decomposition of Plant Litter

The quantity and quality of detritus entering the soil determines the rate of decomposition by microbial communities as well as recycle rates of nitrogen (N) and carbon (C) sequestration1,2. Plant litter comprises the majority of detritus3, and so it is assumed that decomposition is only marginally influenced by biomass inputs from animals such as herbivores and carnivores4,5. However, carnivores may influence microbial decomposition of plant litter via a chain of interactions in which predation risk alters the physiology of their herbivore prey that in turn alters soil microbial functioning when the herbivore carcasses are decomposed6. A physiological stress response by herbivores to the risk of predation can change the C:N elemental composition of herbivore biomass7,8,9 because stress from predation risk increases herbivore basal energy demands that in nutrient-limited systems forces herbivores to shift their consumption from N-rich resources to support growth and reproduction to C-rich carbohydrate resources to support heightened metabolism6. Herbivores have limited ability to store excess nutrients, so stressed herbivores excrete N as they increase carbohydrate-C consumption7. Ultimately, prey stressed by predation risk increase their body C:N ratio7,10, making them poorer quality resources for the soil microbial pool likely due to lower availability of labile N for microbial enzyme production6. Thus, decomposition of carcasses of stressed herbivores has a priming effect on the functioning of microbial communities that decreases subsequent ability to of microbes to decompose plant litter6,10,11.
We present the methodology to evaluate linkages between predation risk and litter decomposition by soil microbes. We describe how to: induce stress in herbivores from predation risk; measure those stress responses, and measure the consequences on microbial decomposition. We use insights from a model grassland ecosystem comprising the hunting spider predator (Pisuarina mira), a dominant grasshopper herbivore (Melanoplus femurrubrum),and a variety of grass and forb plants9.

We present methods to evaluate how predation risk can alter the chemical quality of herbivore prey by inducing dietary changes to meet demands of heightened stress, and how the decomposition of carcasses from these stressed herbivores slows subsequent plant litter decomposition by soil microbes.

Predation 위험, Herbivore 생리적 스트레스와 식물 돼지 미생물 분해를 연결

The quantity and quality of detritus entering the soil  determines the rate of decomposition by microbial communities as well as  recycle rates of ...

Reporter-based Growth Assay for Systematic Analysis of Protein Degradation

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard approach to determining intracellular protein degradation relies on biochemical assays for following the kinetics of protein decline. Such methods are often laborious and time consuming and therefore not amenable to experiments aimed at assessing multiple substrates and degradation conditions. As an alternative, cell growth-based assays have been developed, that are, in their conventional format, end-point assays that cannot quantitatively determine relative changes in protein levels.
Here we describe a method that faithfully determines changes in protein degradation rates by coupling them to yeast cell-growth kinetics. The method is based on an established selection system where uracil auxotrophy of URA3-deleted yeast cells is rescued by an exogenously expressed reporter protein, comprised of a fusion between the essential URA3 gene and a degradation determinant (degron). The reporter protein is designed so that its synthesis rate is constant whilst its degradation rate is determined by the degron. As cell growth in uracil-deficient medium is proportional to the relative levels of Ura3, growth kinetics are entirely dependent on the reporter protein degradation.
This method accurately measures changes in intracellular protein degradation kinetics. It was applied to: (a) Assessing the relative contribution of known ubiquitin-conjugating factors to proteolysis (b) E2 conjugating enzyme structure-function analyses (c) Identification and characterization of novel degrons. Application of the degron-URA3-based system transcends the protein degradation field, as it can also be adapted to monitoring changes of protein levels associated with functions of other cellular pathways.

Here we describe a robust biological assay for quantifying the relative rate of proteolysis by the ubiquitin-proteasome system. The assay readout is yeast growth rate in liquid culture, which is dependent on the cellular levels of a reporter protein comprising a degradation signal fused to an essential metabolic marker.

단백질 분해의 체계적인 분석을위한 기자 기반의 성장 분석

Protein degradation by the ubiquitin-proteasome system (UPS) is a major regulatory mechanism for protein homeostasis in all eukaryotes. The standard ...

Quantitative Immunofluorescence Assay to Measure the Variation in Protein Levels at Centrosomes

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to facilitate sensory functions in cells. The level of a protein may be regulated differently at centrosomes than at other .cellular locations, and the variation in the centrosomal level of several proteins at different points of the cell cycle appears to be crucial for the proper regulation of centriole assembly. We developed a quantitative fluorescence microscopy assay that measures relative changes in the level of a protein at centrosomes in fixed cells from different samples, such as at different phases of the cell cycle or after treatment with various reagents. The principle of this assay lies in measuring the background corrected fluorescent intensity corresponding to a protein at a small region, and normalize that measurement against the same for another protein that does not vary under the chosen experimental condition. Utilizing this assay in combination with BrdU pulse and chase strategy to study unperturbed cell cycles, we have quantitatively validated our recent observation that the centrosomal pool of VDAC3 is regulated at centrosomes during the cell cycle, likely by proteasome-mediated degradation specifically at centrosomes.

Here, a novel quantitative fluorescence assay is developed to measure changes in the level of a protein specifically at centrosomes by normalizing that protein&#8217;s fluorescence intensity to that of an appropriate internal standard.

정량 면역 형광 분석은 중심체에서 단백질 수준의 변화를 측정하기 위해

Centrosomes are small but important organelles that serve as the poles of mitotic spindle to maintain genomic integrity or assemble primary cilia to ...

Electrophoretic Mobility Shift Assay (EMSA) for the Study of RNA-Protein Interactions: The IRE/IRP Example

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron regulatory proteins, IRP1 and IRP2. They bind to iron responsive elements (IREs) within the untranslated regions (UTRs) of several target mRNAs, thereby controlling the mRNAs translation or stability. IRE/IRP interactions have been widely studied by EMSA. Here, we describe the EMSA protocol for analyzing the IRE-binding activity of IRP1 and IRP2, which can be generalized to assess the activity of other RNA-binding proteins as well. A crude protein lysate containing an RNA-binding protein, or a purified preparation of this protein, is incubated with an excess of32 P-labeled RNA probe, allowing for complex formation. Heparin is added to preclude non-specific protein to probe binding. Subsequently, the mixture is analyzed by non-denaturing electrophoresis on a polyacrylamide gel. The free probe migrates fast, while the RNA/protein complex exhibits retarded mobility; hence, the procedure is also called &#8220;gel retardation&#8221; or &#8220;bandshift&#8221; assay. After completion of the electrophoresis, the gel is dried and RNA/protein complexes, as well as free probe, are detected by autoradiography. The overall goal of the protocol is to detect and quantify IRE/IRP and other RNA/protein interactions. Moreover, EMSA can also be used to determine specificity, binding affinity, and stoichiometry of the RNA/protein interaction under investigation.

Electrophoretic Mobility Shift Assay EMSA for the Study of RNA-Protein Interactions: The IRE/IRP Example

Here we present a protocol to analyze RNA/protein interactions. The electrophoretic mobility shift assay (EMSA) is based on the differential migration of RNA/protein complexes and free RNA during native gel electrophoresis. By using a radiolabeled RNA probe, RNA/protein complexes can be visualized by autoradiography.

IRE / IRP 예 : RNA - 단백질 상호 작용의 연구에 대한 전기 이동 기동성 교대 분석 실험 (EMSA)

RNA/protein interactions are critical for post-transcriptional regulatory pathways. Among the best-characterized cytosolic RNA-binding proteins are iron ...