분석을 시작하기 전에, 우리는 다음의 시약이 올바르게 준비되었는지 확인해야합니다 : <ol><li> 50 MM의 칼륨 인산 버퍼, 산도 7.5. 11.4 MG / 칼륨 phospate 이염, 물을 정화하고 1M HCL과 산도를 조정에 trihydrate의 ML을 사용하여 준비합니다. 이 솔루션은 사용하기 전에 ° C 37 배치됩니다. </li><li> 50 MM의 칼륨 인산 버퍼에 카제인의 6.5 MG / ML을 혼합하여 준비 0.65 % 중량 / 부피 카제인 솔루션. 점차적 ° C 10 분 정도 동질적인 분산이 이루어 때까지 80-85로 부드러운 감동과 솔루션 온도를 증가. 이 솔루션을 끓여서하지 않는 것이 매우 중요합니다. NaOH와 HCL와 함께 필요한 경우 산도 그런 다음 조정됩니다. </li><li> 물을 정화와 6.1N 재고 1시 55분을 diluting하여 준비 110 MM Trichloroacetic 산성 용액. Trichloroacetic의 산성이 강한 산성이며 유의해서 다루어 져야합니다. </li><li> 0.5 MM Folin &amp; Ciolcaltea의, 또는 직접적으로 프로 테아제의 활동과 관련이있을 것입니다 측정 색 변화를 생성하는 티로신과 반응 솔루션입니다 Folin의 페놀 시약. Folin의 페놀 시약은 산성이며 유의해서 다루어 져야합니다. </li><li> 물을 정화에 anyhydrous 나트륨 탄산의 53 MG / ML을 사용 준비 500 MM 나트륨 탄산 솔루션. 37 5mM 칼슘 아세테이트 10 MM의 아세트산 나트륨 버퍼, 산도 7.5의 효소로 구성되어 희석제 솔루션 ° C. 이 솔루션은 우리가 고체 테아제 샘플을 분해 또는 효소 솔루션을 희석하는 데 사용하는 어떤 것입니다. </li><li> 1.1 MM L - 티로신 표준 재고 솔루션입니다. 물을 정화하고 티로신이 해소 때까지 부드럽게 온수 0.2 MG / ML L - 티로신을 사용하여 준비 하였다. 카제인과 마찬가지로이 솔루션을 끓여하지 않습니다. L - 티로신 표준 실온으로 냉각하도록 허용합니다. 이 솔루션은 표준 곡선을 만들기 위해 더욱 희석됩니다. </li><li> 단백 분해 효소 솔루션입니다. 사용하기 전에 즉시, 6 단계에서 준비 효소 희석제 솔루션 테아제를 해산. </li></ol> 필요한 경우, 0.1-0.2 단위 / ML하는 효소 희석액을 사용하여 해산합니다 예정된 활동의 견고한 프로 테아제 샘플을 사용합니다. 이 솔루션은 품질 관리 분석에 대한 긍정적인 제어로 우리가 효소 활동을 결정하기 위해 수행되는 계산에 대한 검증 역할을합니다. 단백 분해 효소 분석 및 표준 곡선 설정 <ol><li> 이 분석을 시작하려면 약 15 MLS를 개최합니다 적절한 튜브를 찾으십시오. 테스트 각 효소에 대한 4 튜브가 필요합니다. 한 병에가 빈로 사용되며, 세 사람은 프로 테아제의 세 dilutions의 분석 활동하는 데 사용됩니다. 서로에 대한 최종 계산을 확인할 때 세 dilutions가 유용합니다. <ol><li> 네 튜브의 각 설정하려면, 우리의 0.65 %의 카제인 솔루션의 5mls를 추가합니다. 그들이 37 물 목욕으로 평형하자 ° C를 약 5 분하십시오. </li><li> 공백이 아닌 테스트 샘플 튜브 세 가지로 테스트됩니다 효소 솔루션의 볼륨을 변화하지만, 추가합니다. ° C 정확히 십분을 휘몰아치는 37에 대한 부화하여 섞는다. 이 배양 시간 동안 프로 테아제의 활동과 티로신의 결과적 해방은 측정 및 테스트 샘플 사이에 비교 될거야. </li></ol></li><li> 이 10 분 부화 후, 반응을 중지 각 튜브에 TCA 시약의 5​​ MLS를 추가합니다. 그런 다음, 각 튜브에 심지어 빈, 그래서 각각의 튜브에 효소 용액의 최종 부피가되는 한 ML을 효소 솔루션의 적절한 볼륨을 추가합니다. 이것은 효소 자체의 흡광도 값을 고려하여 각 튜브의 최종 볼륨이 동일한지 확인하는 일을합니다. 37 솔루션을 품어 ° C를 30 분. 이 30 분 동안 부화, 당신은 티로신 표준 dilutions을 설정할 수 있습니다. 쉽게 팔 MLS를 저장할 수있는 6 DRAM 튜브 (DRAM의 튜브가 폴리 프로필렌 튜브로 대체 수 있습니다)를 사용합니다. 0.05, 0.10, 0.20, 0.40, 0.50 : 여섯 튜브하려면, MLS에서 다음 볼륨으로 1.1 MM 티로신 표준 재고 솔루션을 추가합니다. 빈 어떤 티로신 표준을 추가하지 마십시오. 낮은 표준은 약간의 색상 변화를 얻을 것입니다 불순한 테스트 샘플에 필요한 수 있습니다. 티로신 표준 솔루션이 추가되면, 2 MLS에 볼륨을 가져 기준 각 정화 물을 적절한 볼륨을 추가합니다. </li><li> 30 분 부화 후, 테스트 솔루션의 각 필터와 빈이 0.45 음 polyethersulfone 주사기 필터를 사용합니다. 여과는 시료로부터 insolubles를 제​​거하는 데 필요합니다. <ol><li> 최소한 8 MLS를 저장할 수있는 4 DRAM의 튜브로 여과 2 테스트 샘플의 MLS와 빈 여과물을 추가합니다. 기준가 준비가되어있는 유리병의 동일한 유형을 사용하실 수 있습니다. </li><li> 기준 및 표준 공백이 들어있는 유리병 모든, 나트륨 탄산염의 5mls를 추가합니다. 최상의 결과를 얻으려면, 즉시 나중에 Folin의 시약 1 ML를 추가합니다. </li><li> Folin의 시약을 추가하여 만든 산도 드롭을 규제하기 위해 나트륨 탄산염을 추가합니다. </li><li> 테스트 샘플 드디어으로 나트륨 탄산 추가t 비워. 이러한 솔루션은 나트륨 탄산염의 추가 후 흐림된다. 무료 티로신과 주로 반응 Folin의 시약을 추가합니다. </li><li> 소용돌이 치는하여 DRAM의 튜브를 섞어 30 분 동안 37 ° C에서 알을 품다. </li></ol> 이 부화 후, 당신은 표준 티로신 추가의 금액과 연관 색상의 그라데이션이 있다고 통지해야한다 티로신의 최고 농도는 어두운 게재. 당신은 또한 우리의 테스트 샘플에서 감지할 수있을 정도의 색상 변경을 통지하실 수 있습니다. 이러한 솔루션 2mls는 적합한 cuvettes에 0.45 음 polyethersulfone 주사기 필터를 사용하여 필터링됩니다. 분석이 수행되는 이제, 당신은 우리의 흡광도 값을 기록 분광 광도계로 진행할 수 있습니다. </li></ol> 흡광도를 측정하고 효소 활동을 계산 <ol><li> 샘플의 흡광도는 660nm의 파장을 사용하여 분광 광도계로 측정됩니다. 조명 경로는 1cm로 설정됩니다. 표준, 표준 빈, 다른 시험 샘플 테스트 비워에 대한 흡광도 값을 기록합니다. 일단 모든 데이터가 수집되어, 표준 곡선을 만들 수 있습니다. 표준 및 표준 빈 사이의 흡광도의 곡선을 생성하기 위해 차이는 계산해야합니다. 이것은 표준 솔루션 티로신의 양에 기인 흡광도 값이다. 이 간단한 계산 후, Y 축에 대한 기준 흡광도의 변화, 대 X 축에 대한 다섯 표준의 각 micromoles에 금액을 계획하는 그래프 프로그램을 사용하여 표준 곡선을 만듭니다. <table style="width: 416px; height: 118px;" border="0"><tbody><tr><th style="text-align:center"> 티로신 표준의 볼륨 </th><th style="text-align:center"> uMoles의 티로신 </th></tr><tr><td style="text-align:center"> 0.05 </td><td style="text-align:center"> 0.055 </td></tr><tr><td style="text-align:center"> 0.10 </td><td style="text-align:center"> 0.111 </td></tr><tr><td style="text-align:center"> 0.20 </td><td style="text-align:center"> 0.221 </td></tr><tr><td style="text-align:center"> 0.40 </td><td style="text-align:center"> 0.442 </td></tr><tr><td style="text-align:center"> 0.50 </td><td style="text-align:center"> 0.553 </td></tr></tbody></table></li><li> 데이터 포인트가 입력되고 나면, 가장 적합한 및 해당 슬로프 방정식의 라인을 생성합니다. 테스트 샘플 흡광도 및 테스트 빈의 흡광도의 차이를 계산하여 테스트 샘플의 흡광도 변화를 찾으십시오. 슬로프 방정식에 테스트 샘플 중 하나에 대한 흡광도 값을 삽입 및 해결하면이 특정 proteolytic 반응 동안 티로신 해방의 micromoles가 발생합니다. / ML 당 단위로 효소의 활동을하려면, 다음과 같은 계산을 수행합니다 : (umole 티로신 등가물이 발표) X (11) 단위 / ML 효소 = __________________________________________ (1) X (10) X (2) 분석 11 = 총 볼륨 (밀리리터)을 분석 10 = 시간 (분)을 단위로 정의 당 한 효소의 효소 = 볼륨 (밀리리터)를 사용 Colorimetric 결정에 사용되는 2 = 볼륨 (밀리리터)을 슬로프 방정식에서 얻은 micromoles 티로신 동일한 릴리스의 개수를 가지고 우리의 경우에는 11mls있다 MLS에서 분석의 총 볼륨에 의해 그것을 곱하면됩니다. 세 가지 다른 수량하여이 값을 나누기 : 우리가 10 분 동안 실행된 분석의 시간, 분석에 사용되는 효소의 볼륨은, (가 1ml를 사용하자) 다양한되었던 colorimetric 검출에 사용 밀리리터의 부피, 어떤 수 있습니다 귀하의 쿠베트에 따라 다릅니다. 우리는 MLS 2를 사용합니다. </li><li> 티로신의 Micromoles는 &quot;단위&quot;라는 단백 분해 효소 활동의 분 수율 측정 시간으로 나눠 계산합니다. 우리는 단위 / ML의 관점에서 효소 활동의 measurment두고, 분자와 분모의 볼륨 측정 단위를 취소할 수 있습니다. 효소 희석액에 희석 솔리드 프로 테아제 샘플의 활동을 결정하기 위해, 우리는 단위 / MG의 관점에서 활동으로 우리를 떠나지에서 MG / ML. 원래이 분석에 사용되는 고체의 농도에 의해 단위 / ML 우리의 활동을 나누어 . 단위 / ML 효소 단위 / MG 고체 = _____________________ MG 솔리드 / ML 효소 </li></ol>

<ol>
	<li>Anson, M. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Gen. Physiol. 22, 79-89 (1938).</li><li>Folin, O., Ciocalteau, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> J. Biol. Chem. 73, 627 (1929).</li></ol>

우리는 시그마의 보편적인 프로 테아제 활동 분석을 사용하는 프로 테아제 활동을 분석하는 방법을 보여했습니다. 또한,이 분석은 실험을 위해 그들을 받게하기 전에 우리 프로 테아제는 정확하게 활동을 결정했는지 확인하는 데 유용합니다. 당신이 본 것처럼,이 절차를 수행할 때, 그것은 천천히 아니라 그들을 끓일 수있는 카제인과 티로신 솔루션을 모두 열을 기억 파라마운트 중요 하군요. 또한, 둘 다 표준과 당신이 가진 각 테스트 샘플에 대해 서로 다른 공백을 준비하는 매우 중요하다.

<table>
	<tbody>
		<tr>
			<td>Protease</td>
			<td>Sigma-Aldrich</td>
			<td>P4630</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium Phosphate, Dibasic, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>P5504</td>
			<td></td>
		</tr>
		<tr>
			<td>Casein</td>
			<td>Sigma-Aldrich</td>
			<td>C7078</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichloroacetic Acid</td>
			<td>Sigma-Aldrich</td>
			<td>T0699</td>
			<td></td>
		</tr>
		<tr>
			<td>Folin &#38; Ciocalteu&#39;s Phenol Reagent</td>
			<td>Sigma-Aldrich</td>
			<td>F9252</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Carbonate, Anhydrous</td>
			<td>Sigma-Aldrich</td>
			<td>S2127</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium Acetate, Trihydrate</td>
			<td>Sigma-Aldrich</td>
			<td>S8625</td>
			<td></td>
		</tr>
		<tr>
			<td>Calcium Acetate</td>
			<td>Sigma-Aldrich</td>
			<td>C1000</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Tyrosine, Free Base</td>
			<td>Sigma-Aldrich</td>
			<td>T3754</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Profiler Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PP0500</td>
			<td></td>
		</tr>
		<tr>
			<td>Protease Fluorescent Detection Kit</td>
			<td>Sigma-Aldrich</td>
			<td>PF0100</td>
			<td></td>
		</tr>
	</tbody>
</table>

sigma's non-specific protease activity assay - casein as a substrate

프로 테아제는 펩티드 채권을 깰. 연구실에서, 그것은 종종 프로 테아제의 활동을 측정 및 / 또는 비교하는 것이 필요합니다. 시그마의 불특정 프로 테아제 활동 분석은 우리의 품질 관리 절차 동안 어떤 일을 프로 테아제의 활동을 결정하는 표준화된 절차로 사용할 수 있습니다. 이 분석에서는 카제인은 기판 역할을합니다. 우리가 테스트하는 프로 테아제가 카제인 소화되면 아미노산 티로신은 다른 아미노산과 펩타이드 조각과 함께 해방됩니다. Folin 및 Ciocalteus 페놀, 또는 Folin의 시약은 주로 같지는와 분광 광도계에 흡광도 값으로 측정하는 파란 색깔의 발색단를 생산하는 무료 티로신과 반응. 카제인에서 릴리스 더 티로신, 더 chromophores이 프로 테아제의 활동을 생성하고 강한입니다. 단백 분해 효소의 활동에 의해 생성된 흡광도 값 micromoles에서 티로신의 양을와 흡광도의 변화를 상호 연관시킬 FC 시약과 티로신 알려진 수량 반응에 의해 생성된 표준 곡선에 비교됩니다. 표준 곡선에서 프로 테아제 샘플의 활동은 분당 카제인에서 출시 티로신 등가물의 micromoles에서 금액 단위의 측면에서 결정 수 있습니다. 중국어로이 문서를 보려면 <a href="https://jove-com.remotexs.ntu.edu.sg/index/details.stp?ID=910">여기를 클릭하십시오</a>

Watch this Scientific Journal Video about Sigma's Non-specific Protease Activity Assay - Casein as a Substrate at JoVE.com

Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

프로 테아제는 펩티드 채권을 깰. 연구실에서, 그것은 종종 프로 테아제의 활동을 측정 및 / 또는 비교하는 것이 필요합니다. 시그마의 불특정 프로 테아제 활동 분석은 프로 테아제의 활동을 결정하는 표준화된 절차로 사용할 수 있습니다.

시그마의 비 특정 프로 테아제의 활동 분석 - 카제인 기판으로

Proteases break peptide bonds. In the lab, it is often necessary to measure and/or compare the activity of proteases.  Sigma's non-specific protease ...

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비디오: Sigma's Non-specific Protease Activity Assay - Casein as a Substrate

Rapid Genotyping of Mouse Tissue Using Sigma's Extract-N-Amp Tissue PCR Kit

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is determined. Conventional preparation of tissues for PCR-ready DNA often take several hours to days, depending on the tissue sample. The genotype of the sample may thus be delayed for several days, which is not an option for many different types of experiments. Here we demonstrate the complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR Kit. First, we demonstrate the fifteen-minute extraction of DNA from the tissue sample. Then, we demonstrate the real time read-out of the PCR amplification of the sample, which allows for the identification of a positive sample as it is being amplified. Together, the rapid extraction and real-time readout allow for a prompt identification of genotype of a variety different types of tissues through the reliable method of PCR.

The complete genotyping of a mouse tail sample, including tissue digestion and PCR readout, is done in one and a half hours using Sigma's SYBR Green Extract-N-Amp Tissue PCR kit.

시그마의 압축을 풉니다 - N - 앰프 조직 PCR 키트를 사용하여 마우스 조직의 신속한 Genotyping

Genomic detection of DNA via PCR amplification and detection on an electrophoretic gel is a standard way that the genotype of a tissue sample is ...

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Use of the Protease Fluorescent Detection Kit to Determine Protease Activity

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect protease activity uses casein labeled with fluorescein isothiocyanate (FITC) as the substrate.
Protease activity results in the cleavage of the FITC-labeled casein substrate into smaller fragments, which do not precipitate under acidic conditions. After incubation of the protease sample and substrate, the reaction is acidified with the addition of trichloroacetic acid (TCA). The mixture is then centrifuged with the undigested substrate forming a pellet and the smaller, acid soluble fragments remaining in solution. The supernatant is neutralized and the fluorescence of the FITC-labeled fragments is measured.
The described kit procedure detects the trypsin protease control at a concentration of approximately 0.5 &mu;g/ml (5 ng of trypsin added to the assay). This sensitivity can be increased with a longer incubation time, up to 24 hours. The assay is performed in microcentrifuge tubes and procedures are provided for fluorescence detection using either cuvettes or multiwell plates.

The Protease Fluorescent Detection Kit is designed for the measurement of protease activity using fluorometry. It is also suitable for detection of trace amounts of protease contamination. The method is based on the proteolytic hydroysis of a proprietary formulation of a FITC-labeled casein substrate.

단백 분해 효소의 활동을 확인하는 프로 테아제 형광 검출 키트의 사용

The Protease Fluorescent Detection Kit provides ready-to-use reagents for detecting the presence of protease activity. This simple assay to detect ...

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

의 활동을 측정하는 어세이 대장균은 라이신 Decarboxyase을 Inducible

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

A Matrigel-Based Tube Formation Assay to Assess the Vasculogenic Activity of Tumor Cells

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic activity of vascular endothelial cells in vitro1-5. However, recently growing evidence has shown that this assay is not limited to test vascular behavior for endothelial cells. Instead, it also has been used to test the ability of a number of tumor cells to develop a vascular phenotype6-8. This capability was consistent with their vasculogenic behavior identified in xenotransplanted animals, a process known as vasculogenic mimicry (VM)9. There is a multitude of evidence demonstrating that tumor cell-mediated VM plays a vital role in the tumor development, independent of endothelial cell angiogenesis6, 10-13. For example, tumor cells were found to participate in the blood perfused, vascular channel formation in tissue samples from melanoma and glioblastoma patients8, 10, 11. Here, we described this tubular network assay as a useful tool in evaluation of vasculogenic activity of tumor cells. We found that some tumor cell lines such as melanoma B16F1 cells, glioblastoma U87 cells, and breast cancer MDA-MB-435 cells are able to form vascular tubules; but some do not such as colon cancer HCT116 cells. Furthermore, this vascular phenotype is dependent on cell numbers plated on the Matrigel. Therefore, this assay may serve as powerful utility to screen the vascular potential of a variety of cell types including vascular cells, tumor cells as well as other cells.

A tube formation assay is used to evaluate vascular activity of tumor cells.

종양 세포의 Vasculogenic 활동을 평가 Matrigel 기반 튜브 형성 분석

Over the past several decades, a tube formation assay using growth factor-reduced Matrigel has been typically employed to demonstrate the angiogenic ...

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a GTPase domain (termed ROC, for Ras of Complex proteins) and a kinase domain1. The discovery in 2004 of mutations in LRRK2 that cause Parkinson's disease (PD) resulted in LRRK2 being the focus of a huge volume of research into its normal function and how the protein goes awry in the disease state2,3. Initial investigations into the function of LRRK2 focused on its enzymatic activities4-6. Although a clear picture has yet to emerge of a consistent alteration in these due to mutations, data from a number of groups has highlighted the importance of the kinase activity of LRRK2 in cell death linked to mutations7,8. Recent publications have reported inhibitors targeting the kinase activity of LRRK2, providing a key experimental tool9-11. In light of these data, it is likely that the enzymatic properties of LRRK2 afford us an important window into the biology of this protein, although whether they are potential drug targets for Parkinson's is open to debate.
A number of different approaches have been used to assay the kinase activity of LRRK2. Initially, assays were carried out using epitope tagged protein overexpressed in mammalian cell lines and immunoprecipitated, with the assays carried out using this protein immobilised on agarose beads4,5,7. Subsequently, purified recombinant fragments of LRRK2 in solution have also been used, for example a GST tagged fragment purified from insect cells containing residues 970 to 2527 of LRRK212. Recently, Dani&euml;ls et al. reported the isolation of full length LRRK2 in solution from human embryonic kidney cells, however this protein is not widely available13. In contrast, the GST fusion truncated form of LRRK2 is commercially available (from Invitrogen, see table 1 for details), and provides a convenient tool for demonstrating an assay for LRRK2 kinase activity. Several different outputs for LRRK2 kinase activity have been reported. Autophosphorylation of LRRK2 itself, phosphorylation of Myelin Basic Protein (MBP) as a generic kinase substrate and phosphorylation of an artificial substrate - dubbed LRRKtide, based upon phosphorylation of threonine 558 in Moesin - have all been used, as have a series of putative physiological substrates including &alpha;-synuclein, Moesin and 4-EBP14-17. The status of these proteins as substrates for LRRK2 remains unclear, and as such the protocol described below will focus on using MBP as a generic substrate, noting the utility of this system to assay LRRK2 kinase activity directed against a range of potential substrates.

Assaying the Kinase Activity of LRRK2 in vitro

Leucine Rich Repeat Kinase 2 is a large multidomain kinase, mutations in which are the most common genetic cause of Parkinson's disease. Analysis of the kinase activity of this protein has proven to be a crucial tool in understanding the biology and dysfunction of this protein. In this paper, in vitro assaying of the kinase activity of LRRK2 and a selection of its mutants is described, providing an experimental system to examine phosphorylation of putative substrates and potential dysfunction of LRRK2 in disease.

LRRK2의 키나제 활동 시금 체외에서</em

Leucine Rich Repeat Kinase 2 (LRRK2) is a 2527 amino acid member of the ROCO family of proteins, possessing a complex, multidomain structure including a ...

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic mechanisms of proteolytic enzymes1-3. With the advent of mass spectrometry-based proteomics, the enzymatically-catalyzed incorporation of 18O-atoms from stable isotopically enriched water has become a popular method to quantitatively compare protein expression levels (reviewed by Fenselau and Yao4, Miyagi and Rao5 and Ye et al.6). 18O-labeling constitutes a simple and low-cost alternative to chemical (e.g. iTRAQ, ICAT) and metabolic (e.g. SILAC) labeling techniques7. Depending on the protease utilized, 18O-labeling can result in the incorporation of up to two 18O-atoms in the C-terminal carboxyl group of the cleavage product3. The labeling reaction can be subdivided into two independent processes, the peptide bond cleavage and the carboxyl oxygen exchange reaction8. In our PALeO (protease-assisted labeling employing 18O-enriched water) adaptation of enzymatic 18O-labeling, we utilized 50% 18O-enriched water to yield distinctive isotope signatures. In combination with high-resolution matrix-assisted laser desorption ionization time-of-flight tandem mass spectrometry (MALDI-TOF/TOF MS/MS), the characteristic isotope envelopes can be used to identify cleavage products with a high level of specificity. We previously have used the PALeO-methodology to detect and characterize endogenous proteases9 and monitor proteolytic reactions10-11. Since PALeO encodes the very essence of the proteolytic cleavage reaction, the experimental setup is simple and biochemical enrichment steps of cleavage products can be circumvented. The PALeO-method can easily be extended to (i) time course experiments that monitor the dynamics of proteolytic cleavage reactions and (ii) the analysis of proteolysis in complex biological samples that represent physiological conditions. PALeO-TimeCourse experiments help identifying rate-limiting processing steps and reaction intermediates in complex proteolytic pathway reactions. Furthermore, the PALeO-reaction allows us to identify proteolytic enzymes such as the serine protease trypsin that is capable to rebind its cleavage products and catalyze the incorporation of a second 18O-atom. Such "double-labeling" enzymes can be used for postdigestion 18O-labeling, in which peptides are exclusively labeled by the carboxyl oxygen exchange reaction. Our third strategy extends labeling employing 18O-enriched water beyond enzymes and uses acidic pH conditions to introduce 18O-stable isotope signatures into peptides.

Protease- and Acid-catalyzed Labeling Workflows Employing 18O-enriched Water

Stable isotope labeling workflows employing 18O-enriched water (LeO-workflows) are versatile tools for quantitative and qualitative proteomics studies. In protease-assisted (PALeO) workflows, 18O-atoms are introduced by proteolytic cleavage and carboxyl oxygen exchange reactions mediated by proteases. In the acid-catalyzed (ALeO) workflow, 18O-atoms are introduced by carboxyl oxygen exchange at low pH.

도입 단백 분해 효소와 산 촉매 라벨링 워크 플로<sup&gt; 18</sup&gt; O 강화 물

Stable isotopes are essential tools in biological mass spectrometry. Historically, 18O-stable isotopes have been extensively used to study the catalytic ...

Mouse Islet of Langerhans Isolation using a Combination of Purified Collagenase and Neutral Protease

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines to the study of isolated rodent islets. Primary islets offer the distinct advantage that they more faithfully reflect the biology of intracellular signaling pathways and secretory responses. Whereas the method of islet isolation using tissue dissociating enzyme (TDE) preparations has been well established in many laboratories1-4, variations in the consistency of islet yield and quality from any given rodent strain limit the extent and feasibility of primary islet studies. These variations often occur as a result of the crude partially purified TDEs used in the islet isolation procedure; TDEs frequently exhibit lot-to-lot variations in activity and often require adjustments to the dose of enzyme used. A small number of reports have used purified TDEs for rodent cell isolations5, 6, but the practice is not widespread despite the routine use and advantages of purified TDEs for human islet isolations. In collaboration with VitaCyte, LLC (Indianapolis, IN), we developed a modified mouse islet isolation protocol based on that described by Gotoh7, 8, in which the TDEs are perfused directly into the pancreatic duct of mice, followed by crude tissue fractionation through a Histopaque gradient9, and isolation of purified islets. A significant difference in our protocol is the use of purified collagenase (CIzyme MA) and neutral protease (CIzyme BP) combination. The collagenase was characterized by the use of a6 fluorescence collagen degrading activity (CDA) assay that utilized fluorescently labeled soluble calf skin fibrils as substrate6. This substrate is more predictive of the kinetics of collagen degradation in the tissue matrix because it relies on native collagen as the substrate. The protease was characterized with a sensitive fluorescent kinetic assay10. Utilizing these improved assays along with more traditional biochemical analysis enable the TDE to be manufactured more consistently, leading to improved performance consistency between lots. The protocol described in here was optimized for maximal islet yield and optimal islet morphology using C57BL/6 mice. During the development of this protocol, several combinations of collagenase and neutral proteases were evaluated at different concentrations, and the final ratio of collagenase:neutral protease of 35:10 represents enzyme performance comparable to Sigma Type XI. Because significant variability in average islet yields from different strains of rats and mice have been reported, additional modifications of the TDE composition should be made to improve the yield and quality of islets recovered from different species and strains.

A detailed description of mouse islet isolation is described using the technique of in situ pancreatic ductal cannulation and perfusion of a combination of purified collagenase and neutral protease.

Langerhans 절연의 마우스 작은 섬은 정화의 Collagenase와 중성 단백 분해 효소의 조합을 사용하여

The interrogation of beta cell gene expression and function in vitro has squarely shifted over the years from the study of rodent tumorigenic cell lines ...

Determining Membrane Protein Topology Using Fluorescence Protease Protection (FPP)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established for many membrane proteins. A simple technique called fluorescence protease protection (FPP) is presented, which permits the determination of membrane protein topology in living cells. This technique has numerous advantages over other methods for determining protein topology, in that it does not require the availability of multiple antibodies against various domains of the membrane protein, does not require large amounts of protein, and can be performed on living cells. The FPP method employs the spatially confined actions of proteases on the degradation of green fluorescent protein (GFP) tagged membrane proteins to determine their membrane topology and orientation. This simple approach is applicable to a wide variety of cell types, and can be used to determine membrane protein orientation in various subcellular organelles such as the mitochondria, Golgi, endoplasmic reticulum and components of the endosomal/recycling system. Membrane proteins, tagged on either the N-termini or C-termini with a GFP fusion, are expressed in a cell of interest, which is subject to selective permeabilization using the detergent digitonin. Digitonin has the ability to permeabilize the plasma membrane, while leaving intracellular organelles intact. GFP moieties exposed to the cytosol can be selectively degraded through the application of protease, whereas GFP moieties present in the lumen of organelles are protected from the protease and remain intact. The FPP assay is straightforward, and results can be obtained rapidly.

Determining Membrane Protein Topology Using Fluorescence Protease Protection FPP

Here, we present a protocol to determine the orientation and topology of integral membrane proteins in living cells. This simple protocol relies on selective protease sensitivity of chimeras between the protein of interest and GFP.

형광 단백질 분해 효소 보호를 사용하여 막 단백질 토폴로지를 결정 (FP​​P)

The correct topology and orientation of integral membrane proteins are essential for their proper function, yet such information has not been established ...

Comet Assay as an Indirect Measure of Systemic Oxidative Stress

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively inert because it has two unpaired electrons located in different pi * anti-bonding orbitals. These two electrons have parallel spins, meaning they rotate in the same direction about their own axes. This is why the oxygen molecule is not very reactive. Activation of oxygen may occur by two different mechanisms; either through reduction via one electron at a time (monovalent reduction), or through the absorption of sufficient energy to reverse the spin of one of the unpaired electrons. This results in the production of reactive oxidative species (ROS). There are a number of ways in which the human body eliminates ROS in its physiological state. If ROS production exceeds the repair capacity, oxidative stress results and damages different molecules. There are many different methods by which oxidative stress can be measured. This manuscript focuses on one of the methods named cell gel electrophoresis, also known as &#8220;comet assay&#8221; which allows measurement of DNA breaks. If all factors known to cause DNA damage, other than oxidative stress are kept constant, the amount of DNA damage measured by comet assay is a good parameter of oxidative stress. The principle is simple and relies upon the fact that DNA molecules are negatively charged. An intact DNA molecule has such a large size that it does not migrate during electrophoresis. DNA breaks, however, if present result in smaller fragments which move in the electrical field towards the anode. Smaller fragments migrate faster. As the fragments have different sizes the final result of the electrophoresis is not a distinct line but rather a continuum with the shape of a comet. The system allows a quantification of the resulting &#8220;comet&#8221; and thus of the DNA breaks in the cell.

Comet assay measures DNA breaks, induced by different factors. If all factors (except oxidative stress) causing DNA damage are kept constant, the amount of DNA damage is a good indirect parameter of oxidative stress. The goal of this protocol is to use comet assay for indirect measurement of oxidative stress.

전신 산화 스트레스의 간접 측정으로 혜성 분석

Higher eukaryotic organisms cannot live without oxygen; yet, paradoxically, oxygen can be harmful to them. The oxygen molecule is chemically relatively ...

Identification of Kinase-substrate Pairs Using High Throughput Screening

We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel signal transduction pathways. Our approach features the use of a library of purified GST-tagged human protein kinases and a recombinant protein substrate of interest. We have used this technology to identify MAP/microtubule affinity-regulating kinase 2 (MARK2) as the kinase for a glucose-regulated site on CREB-Regulated Transcriptional Coactivator 2 (CRTC2), a protein required for beta cell proliferation, as well as the Axl family of tyrosine kinases as regulators of cell metastasis by phosphorylation of the adaptor protein ELMO. We describe this technology and discuss how it can help to establish a comprehensive map of how cells respond to environmental stimuli.

Protein phosphorylation is a central feature of how cells interpret and respond to information in their extracellular milieu. Here, we present a high throughput screening protocol using kinases purified from mammalian cells to rapidly identify kinases that phosphorylate a substrate(s) of interest.

높은 처리량 검열을 사용 키나제 기판 쌍의 식별


We have developed a screening platform to identify dedicated human protein kinases for phosphorylated substrates which can be used to elucidate novel ...