남동 시작 자금에서 노스 캐롤라이나 대학, 뿐만 아니라 듀크 기부 특별 한 이니셔티브 기금, 웰 스 파고 기금, PhRMA 재단에서 지원 샬 롯을 인정합니다.

1입니다. 복잡 한 BSA-Au(III)의 합성
<ol><li>고성능 액체 크로마토그래피 (HPLC) 학년 물 5 mL 반응 유리병에서의 1 mL에 BSA의 25 mg을 디졸브. 참고: 솔루션 분명 나타납니다.</li>
	<li>HPLC 학년 물에 5 m m의 농도에 금 (III) 염화 물 안내 (chloroauric 산)을 분해. 참고: 솔루션 노란색 나타납니다. Chloroauric 산 성 솔루션이이 농도에서 준비는 BSA Au 비율 1시 13분의 발생 합니다.<ol>
<li>어디의 농도와 chloroauric 산의 솔루션을 준비 또는 0.38 m m 사이 (BSA:Au = 1:1) 20 m m (BSA:Au = 1시 50분) HPLC에 학년 물. 참고: 금 BSA의 다른 비율 여기 방출 지도 크게 다른 빨간 형광 패턴에 발생 합니다.</li>
	</ol>
</li>
	<li>37 ° C 물 욕조에 BSA의 반응 유리병을 배치 하 고 적극적으로 자력을 사용 하 여 750 rpm에서 저 어.</li>
	<li>교 반 시작, 후에 즉시 솔루션에 chloroauric 산의 1 mL를 추가 합니다. 솔루션의 색 노란색 클리어에서 변환 해야 합니다.</li>
	<li>37 ° C에서와 750 rpm는 자력을 사용 하 여 2 분 동안 혼합물을 저 어.</li>
	<li>반응 유리병으로 1 M NaOH의 100 μ 12 pH를가지고 솔루션을 추가 합니다. 참고: NaOH를 추가 후에 즉시 솔루션 노란색-갈색으로 약간 어둡게 한 해야 다음 다시 노란색으로 설정 합니다.</li>
	<li>2 h 750 rpm에서 고 37 ° c.에 저 어로 진행 솔루션 천천히 어두운 노랑/브라운 색상을 노란색에서 변경 해야 합니다. 이 색깔 변화 복잡 한 붉은 fluorescing BSA-Au(III)의 형성을 나타냅니다.</li>
	<li>2 일 동안 실내 온도에 앉아 샘플을 허용 하 고 솔루션 황색 갈색 어둡게 하 계속 되 고 형광 강도 증가.<ol>
<li>또는, 샘플 솔루션의 컬러는 주황색 갈색에 발전 함에 따라 더 많은 h 12 37 ° C에서 저를 계속 하자.</li>
	</ol>
</li>
</ol>2입니다. BSA-Cu(II)-Au(III)의 합성
<ol><li>HPLC 학년 물의 1 mL에서 BSA의 25 mg을 디졸브. 솔루션은 분명 나타납니다.</li>
	<li>HPLC 학년 물 5 m m의 농도에 구리 (II) 염화 물이 수화물을 디졸브. 솔루션 빛 나타납니다 파란색.</li>
	<li>5 mL 반응 유리병을 BSA 용액 1 mL를 추가 하 고 37 ° c.에 물 욕조에 배치 750 rpm에 혼합물을 저 어.</li>
	<li>즉시 반응 유리병에 구리 (II) 염화 물이 수화물 해결책의 0.5 mL을 추가 하 고 2 분에 대 한 믹스. 솔루션 빛 남아 블루.</li>
	<li>하는 pH 12 2 h에 대 한 혼합 수 1 M NaOH의 75 μ를 추가 합니다. 솔루션은 보라색 될 것입니다.</li>
	<li>HPLC 학년 물 5 m m의 농도에 chloroauric 산을 분해.</li>
	<li>반응 유리병에 수성 chloroauric 산의 0.5 mL을 추가 하 고 다시 1 M NaOH를 사용 하 여 12에 pH를 조정.</li>
	<li>2 시간에 대 한 반응 혼합물을 저 어. 참고: 솔루션 갈색 색상으로 진화 한다.</li>
</ol>3입니다. BSA-Ni(II)-Au(III)의 합성
<ol><li>HPLC 학년 물의 1 mL에서 BSA의 25 mg을 디졸브. 솔루션은 분명 나타납니다.</li>
	<li>니켈 (II) 염화 물 hexahydrate 5 m m의 농도를 HPLC 학년 물에 용 해. 솔루션 나타납니다 빛 녹색.</li>
	<li>5 mL 반응 유리병에 BSA 용액 1 mL를 추가 하 고 750 rpm에 혼합물 37 oC. 볶음에 물 욕조에 배치.</li>
	<li>즉시 반응 유리병을 2 분 동안 혼합 니켈 (II) 염화 물 hexahydrate 솔루션의 0.5 mL를 추가 합니다. 참고: 솔루션 빛 유지 됩니다 녹색.</li>
	<li>하는 pH 12 2 h에 대 한 혼합 수 1 M NaOH의 75 μ를 추가 합니다. 참고: 솔루션 어두운 노란색 될 것입니다.</li>
	<li>HPLC 학년 물 5 m m의 농도에 chloroauric 산을 분해.</li>
	<li>반응 유리병에 수성 chloroauric 산의 0.5 mL을 추가 하 고 pH 12 다시 조정 합니다.</li>
	<li>2 시간에 대 한 반응 혼합물을 저 어. 참고: 솔루션 갈색 색상으로 진화 한다.</li>
</ol>4입니다. [Cys34-capped-BSA]-Au(III)의 합성
<ol><li>버퍼링 하는 인산 염 (PBS, pH 7.4)의 1 mL에 N-ethylmaleimide (NEM)의 2 밀리 그램을 디졸브.</li>
	<li>1 mL의 PBS NEM 솔루션에 BSA의 2 밀리 그램을 디졸브.</li>
	<li>5 mL 반응 유리병에 솔루션을 전송 하 고 1 시간에 500 rpm에서 20 ° C에서 저 어.</li>
	<li>500 mL PBS, unreacted NEM 제거를 자력 하룻밤 함께 50 rpm에서 교 반에 12 kDa 투 석 튜브를 사용 하 여 솔루션 dialyze</li>
	<li>0.4 m m의 농도에 PBS에 chloroauric 산을 분해. 참고: 솔루션 희미 한 노란색을 될 것입니다.</li>
	<li>37 ° c.에 물 목욕을 반응 유리병을 전송 750 rpm에서 저 어.</li>
	<li>즉시 반응 유리병에 chloroauric 산 성 해결책의 1 mL을 추가 하 고 2 분 대 혼합을 허용.</li>
	<li>PH 12를가지고 고 2 h에 대 한 혼합 반응 유리병에 1 M NaOH의 75 μ를 추가 합니다.</li>
</ol>5입니다. [all-thiol-capped-BSA]-Au(III)의 합성
<ol><li>2 M 요소와 50 m m 염화 중 탄산염 (NH4HCO3, pH 8.0) HPLC 학년 물에의 한 솔루션을 준비 합니다.</li>
	<li>위의 솔루션의 5 mL 반응 유리병에 1 mL에 BSA의 3.3 mg을 디졸브.</li>
	<li>0.25 M의 재고 솔루션 tris(2-carboxyethyl) HPLC 물의 1 mL에서의 TCEP 62.5 mg를 용 해 하 여 phosphine (TCEP).</li>
	<li>TCEP의 최종 농도 8 m m까지 재고 TCEP 솔루션 반응 유리병에 추가 합니다.</li>
	<li>50 ° c.에 1 시간을 위한 물 욕조에 솔루션을 품 어 자력 교 반기를 사용 하 여 500 rpm에서 저 어.</li>
	<li>실내 온도에 완전히 냉각 솔루션을 수 있습니다.</li>
	<li>12.5 mg NEM의 HPLC 학년 물 1 mL에 용 해 하 여 100mm NEM의 재고 솔루션을 준비 합니다.</li>
	<li>NEM의 최종 농도 16 m m까지 반응 유리병에 NEM의 재고 솔루션을 추가 합니다.</li>
	<li>솔루션 2 h 20 ° c.에 대 한 결합을 허용합니다 500 rpm에서 저 어.</li>
	<li>초과 TCEP, NEM, 및 요소를 제거 하는 50mm NH4HCO3 의 500 mL에서 하룻밤 자력으로 50 rpm에서 솔루션을 저 어 12 kDa 투 석 튜브를 사용 하 여 솔루션 dialyze</li>
	<li>37 ° C에 물 목욕을 반응 유리병을 이동 하 고 750 rpm에서 저 어.</li>
	<li>0.66 m m의 농도를 HPLC 학년 물 산 성 chloroauric를 분해.</li>
	<li>즉시 반응 유리병에 chloroauric 산 성 해결책의 1 mL을 추가 하 고 2 분 대 혼합을 허용.</li>
	<li>해결책의 pH는 12까지 1 M NaOH를 추가 하 고 2 시간에 대 한 혼합 계속 솔루션을 허용.</li>
</ol>

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A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Copper-Binding+Properties+of+Bovine+Serum+Albumin+and+Its+Amino-terminal+Peptide+Fragment.">Copper-Binding Properties of Bovine Serum Albumin and Its Amino-terminal Peptide Fragment.</a> Journal of Biological Chemistry. 242, 1574-1578 (1967).</li><li>Xue, Y., Li, X., Li, H., Zhang, W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantifying+Thiol-Gold+Interactions+towards+the+Efficient+Strength+Control.">Quantifying Thiol-Gold Interactions towards the Efficient Strength Control.</a> Nature Communications. 5, 4348 (2014).</li><li>Xu, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Protein+Characteristics+in+the+Formation+and+Fluorescence+of+Au+Nanoclusters.">The Role of Protein Characteristics in the Formation and Fluorescence of Au Nanoclusters.</a> Nanoscale. 6 (3), 1515-1524 (2014).</li></ol>

저자는 공개 없다.

PH 12에서 BSA-Au(III) 화합물 λ그들의 방출 파장에 빨간색 형광을 전시 = 640 nm로 자외선 (UV) 빛 λ전= 365 nm (그림 1A). 빨간 형광의 출현은 느린 과정이 고 최대 강도를 높이기 위해 실내 온도에 몇 일을 걸릴 것입니다. 37 ° C에서 반응 실행 높은 온도 빨리 빨간 형광을 생산 하 사용할 수 있습니다 최적의 결과 얻을 것 이다. 단백질의 돌이킬 수 없는 저하<sup c...

BSA-Au 화합물 전시는 자외선 (UV)-놀라운와 고르기 빨간색 형광 shift 스톡 스, 시 외원래 합성. 1. 고유 하 고 고정적 빨간 형광2,3,4,5,,67이미징 또는 nanomedicine8 감지 분야에서 다양 한 응용 프로그램을 찾을 수 있습니다 ,,910,11,,1213. 이 화합물은 최근 몇 년 동안14,,1516나노 과학의 분야에서 많은 연구자에 의해 광범위 하 게 공부 되었습니다 했다. BSA-Au 화합물 Au25 nanoclusters로 해석 되었습니다. 제시 방법의 목표는 세부에서이 화합물을 확인 하 고 빨간 형광의 기원 이해를 이다. 제시 방법에 따라, 여러 Au 바인딩 사이트의 존재 그리고 형광, Au25 nanoclusters의 단일 사이트 nucleation 대안의 기원 그림 될 수 있습니다. 동일한 접근은 다른 단백질17,,1819 complexed Au(III)와 그들의 본질적인 형광 속성을 변경할 수 있습니다 어떻게 연구 하 사용할 수 있습니다.
레드-형광 BSA Au 화합물의 합성에는 au (BSA:Au) 여기 방출 지도 (EEM)20에 봉우리의 위치는 형광의 강도 극대화 하는 BSA의 어 금 니 비율의 좁은 제어를 필요 합니다. 그것은 수 표시 여러 바인딩 사이트에 바인딩하려면 Au(III)에 대 한 존재는 아스파라긴 조각 (또는 Asp 조각, BSA의 N-말단에서 처음 4 개의 아미노산 잔류물)를 포함 하 여21,22. BSA (Cys-34)의 34번째 아미노산 Au(III)를 조정 하 고 레드 fluorescence([Cys34-capped-BSA]-Au(III))20의 구조에 참여 하는 것에 표시 됩니다. 모든 Cys Cys 이황화 결합을 고착 하 고 모든 thiols, 빨간색 형광 상한 시은 생산된 ([all-thiol-capped-BSA]-Au(III)). 이 빨간 형광을 생산 하는 Au(III) 바인딩 사이트 Cys Cys 이황화 결합의 필요성을 나타냅니다.
단백질 화학 기법은 하지 널리 나노 과학 커뮤니티에서 BSA-Au(III) 단지 공부를 사용 되었습니다. 그러나, 그것은이 단지, 또한 BSA에서 Au(III) 바인딩 사이트의 상세한 이해로 특정 측면을 이해 하는이 기법을 채택 하는 귀중 한 것입니다. 이 문서는 이러한 기술 중 일부를 보여 것입니다.

<table>
	<tbody>
		<tr>
			<td>Bovine Serum Albumin (BSA), 96%</td>
			<td>Sigma-Aldrich</td>
			<td>A5611</td>
			<td></td>
		</tr>
		<tr>
			<td>gold (III) chloride trihydrate, 99.9%</td>
			<td>Sigma-Aldrich</td>
			<td>520918</td>
			<td></td>
		</tr>
		<tr>
			<td>Copper (II) chloride dihydrate, 99.999%</td>
			<td>Sigma-Aldrich</td>
			<td>459097</td>
			<td></td>
		</tr>
		<tr>
			<td>Nickel (II) chloride hexahydrate, 99.9%</td>
			<td>Sigma-Aldrich</td>
			<td>654507</td>
			<td></td>
		</tr>
		<tr>
			<td>N-Ethylmaleimide (NEM), &#62;99.0%</td>
			<td>Sigma-Aldrich</td>
			<td>4259</td>
			<td></td>
		</tr>
		<tr>
			<td>Tris(2-carboxyethyl)phosphine hydrochloride (TCEP), &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>C4706</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, &#62;98.0%</td>
			<td>Sigma-Aldrich</td>
			<td>S8045</td>
			<td></td>
		</tr>
		<tr>
			<td>Urea, 99.5%</td>
			<td>Chem-Implex Int&#39;l</td>
			<td>30142</td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate buffered saline (PBS)</td>
			<td>Corning</td>
			<td>MT21040CV</td>
			<td></td>
		</tr>
		<tr>
			<td>Ammonium bicarbonate, 99.5%</td>
			<td>Sigma-Aldrich</td>
			<td>9830</td>
			<td></td>
		</tr>
	</tbody>
</table>

luminophore formation in various conformations of bovine serum albumin by binding of gold(iii)

제시 프로토콜의 목적은 Au(III) 바인딩할 구조 변화 유도 빨간색 형광 저조한 BSA의 과정을 공부 하는 (λem = 640 nm) BSA-Au(III) 복합물의. 메서드를 사용 하면 pH 빨간색 형광의 출현을 BSA conformations의 pH 유도 평형 전환 연관 표시를 조정 합니다. 빨간 형광 BSA-Au(III) 단지는 pH 9.7, 이상 해당 BSA의 "양식" 나 란 하의 조정만 형성 수 있습니다. 누구나 어 금 니 비율을 BSA를 조정 하 고 Au(III) 바인딩 과정의 시간 과정을 모니터를 프로토콜 설명 되어 있습니다. 빨간 형광을 생산 하기 위해 BSA 당 Au(III)의 최소 수 미만 7 이다. 우리는 BSA에 여러 Au(III) 바인딩 사이트의 존재를 설명 하는 단계에서 프로토콜을 설명 합니다. 구리를 추가 하 여 첫 번째 (Cu(II)) 또는 니켈 (Ni(II)) 양이온 Au(III) 다음,이 방법을 보여준다 바인딩 사이트 빨간 fluorophore는 Au(III)에 대 한. 둘째, thiol 에이전트를 상한 하 여 BSA를 수정 하 여 다른 nonfluorophore 형성 Au(III) 바인딩 사이트가 드러났습니다. 셋째, 가능한 Au(III) 바인딩 사이트 고착 및 이황화 결합의 상한 BSA 구조 변경, 그림은. 프로토콜 설명, BSA conformations 및 Au(III) 바인딩을 제어 하는 다른 단백질 및 금속 양이온의 상호 작용을 공부 하 일반적으로 적용할 수 있습니다.

BSA-Au(III) 복합물의 형광에서 관찰 되었다 하는 BSA의 본질적인 파란 형광의 변환 (λem = 400 nm) 빨간색 형광을 (λem = 640 nm) 평형 통해 pH 9.7에 대해 발생 합니다 전환 (그림 1)입니다. EEM BSA-Au(III) Au 어 금 니 비율에 다른 BSA에 그림 2에 표시 하 고이 데이터 어떻게 다른 여기 파장에서 같은 방출 파장을 생...

Watch this Scientific Journal Video about Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII at JoVE.com

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

골드 양이온 (소 혈 청 알 부 민 (BSA) 구조적 종속 독특한 BSA Au 형광 특성화에 관해서는 잘 제시의 다양 한 conformations 하 Au(III))의 바인딩 공부에 대 한 프로토콜.

Gold(III)의 바인딩에 의해 소 혈 청 알 부 민의 다양 한 Conformations에 Luminophore 형성

The purpose of the presented protocols is to study the process of Au(III) binding to BSA, yielding conformation change-induced red fluorescence (&#955;em ...

luminophore-formation-various-conformations-bovine-serum-albumin

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JoVE Journal

Chemistry

Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of Gold(III)

6.8K 조회수.  University of North Carolina. 골드 양이온 (소 혈 청 알 부 민 (BSA) 구조적 종속 독특한 BSA Au 형광 특성화에 관해서는 잘 제시의 다양 한 conformations 하 Au(III))의 바인딩 공부에 대 한 프로토콜.

비디오: Luminophore Formation in Various Conformations of Bovine Serum Albumin by Binding of GoldIII

LabVIEW-operated Novel Nanoliter Osmometer for Ice Binding Protein Investigations

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition proteins, are found in cold-adapted organisms and protect them from freeze injuries by interacting with ice crystals. IBPs are found in a variety of organism, including fish1, plants2, 3, arthropods4, 5, fungi6, and bacteria7. IBPs adsorb to the surfaces of ice crystals and prevent water molecules from joining the ice lattice at the IBP adsorption location. Ice that grows on the crystal surface between the adsorbed IBPs develops a high curvature that lowers the temperature at which the ice crystals grow, a phenomenon referred to as the Gibbs-Thomson effect. This depression creates a gap (thermal hysteresis, TH) between the melting point and the nonequilibrium freezing point, within which ice growth is arrested8-10, see Figure 1. One of the main tools used in IBP research is the nanoliter osmometer, which facilitates measurements of the TH activities of IBP solutions. Nanoliter osmometers, such as the Clifton instrument (Clifton Technical Physics, Hartford, NY,) and Otago instrument (Otago Osmometers, Dunedin, New Zealand), were designed to measure the osmolarity of a solution by measuring the melting point depression of droplets with nanoliter volumes. These devices were used to measure the osmolarities of biological samples, such as tears11, and were found to be useful in IBP research. Manual control over these nanoliter osmometers limited the experimental possibilities. Temperature rate changes could not be controlled reliably, the temperature range of the Clifton instrument was limited to 4,000 mOsmol (about -7.5 &deg;C), and temperature recordings as a function of time were not an available option for these instruments.
We designed a custom-made computer-controlled nanoliter osmometer system using a LabVIEW platform (National Instruments). The cold stage, described previously9, 10, contains a metal block through which water circulates, thereby functioning as a heat sink, see Figure 2. Attached to this block are thermoelectric coolers that may be driven using a commercial temperature controller that can be controlled via LabVIEW modules, see Figure 3. Further details are provided below. The major advantage of this system is its sensitive temperature control, see Figure 4. Automated temperature control permits the coordination of a fixed temperature ramp with a video microscopy output containing additional experimental details.
To study the time dependence of the TH activity, we tested a 58 kDa hyperactive IBP from the Antarctic bacterium Marinomonas primoryensis (MpIBP)12. This protein was tagged with enhanced green fluorescence proteins (eGFP) in a construct developed by Peter Davies' group (Queens University)10. We showed that the temperature change profile affected the TH activity. Excellent control over the temperature profile in these experiments significantly improved the TH measurements. The nanoliter osmometer additionally allowed us to test the recrystallization inhibition of IBPs5, 13. In general, recrystallization is a phenomenon in which large crystals grow larger at the expense of small crystals. IBPs efficiently inhibit recrystallization, even at low concentrations14, 15. We used our LabVIEW-controlled osmometer to quantitatively follow the recrystallization of ice and to enforce a constant ice fraction using simultaneous real-time video analysis of the images and temperature feedback from the sample chamber13. The real-time calculations offer additional control options during an experimental procedure. A stage for an inverted microscope was developed to accommodate temperature-controlled microfluidic devices, which will be described elsewhere16.
The Cold Stage System
The cold stage assembly (Figure 2) consists of a set of thermoelectric coolers that cool a copper plate. Heat is removed from the stage by flowing cold water through a closed compartment under the thermoelectric coolers. A 4 mm diameter hole in the middle of the copper plate serves as a viewing window. A 1 mm diameter in-plane hole was drilled to fit the thermistor. A custom-made copper disc (7 mm in diameter) with several holes (500 &mu;m in diameter) was placed on the copper plate and aligned with the viewing window. Air was pumped at a flow rate of 35 ml/sec and dried using Drierite (W.A. Hammond). The dry air was used to ensure a dry environment at the cooling stage. The stage was connected via a 9 pin connection outlet to a temperature controller (Model 3040 or 3150, Newport Corporation, Irvine, California, US). The temperature controller was connected via a cable to a computer GPIB-PCI card (National instruments, Austin, Texas, USA).

Ice binding proteins (IBPs), also known as antifreeze proteins, inhibit ice growth and are a promising additive for use in the cryopreservation of tissues. The main tool used to investigate IBPs is the nanoliter osmometer. We developed a home-designed cooling stage mounted on an optical microscope and controlled using a custom-built LabVIEW routine. The nanoliter osmometer described here manipulated the sample temperature in an ultra-sensitive manner.

아이스 바인딩 단백질 수사 LabVIEW 운영하는 소설 Nanoliter의 Osmometer

Ice-binding proteins (IBPs), including antifreeze proteins, ice structuring proteins, thermal hysteresis proteins, and ice recrystallization inhibition ...

연구

화학

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

울트라 성능 액체 크로마토 그래피 - 질량 분석법 (UPLC-MS)에 의해 혈청 대규모 비 타겟 Metabolomic 프로파일

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Covalent Binding of BMP-2 on Surfaces Using a Self-assembled Monolayer Approach

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell differentiation into osteoblasts, thus stimulating healing and de novo bone formation. The clinical use of recombinant human BMP-2 (rhBMP-2) in conjunction with scaffolds has raised recent controversies, based on the mode of presentation and the amount to be delivered. The protocol presented here provides a simple and efficient way to deliver BMP-2 for in vitro studies on cells. We describe how to form a self-assembled monolayer consisting of a heterobifunctional linker, and show the subsequent binding step to obtain covalent immobilization of rhBMP-2. With this approach it is possible to achieve a sustained presentation of BMP-2 while maintaining the biological activity of the protein. In fact, the surface immobilization of BMP-2 allows targeted investigations by preventing unspecific adsorption, while reducing the amount of growth factor and, most notably, hindering uncontrolled release from the surface. Both short- and long-term signaling events triggered by BMP-2 are taking place when cells are exposed to surfaces presenting covalently immobilized rhBMP-2, making this approach suitable for in vitro studies on cell responses to BMP-2 stimulation.

We describe a method for accomplishing efficient immobilization of BMP-2 on surfaces. Our approach is based on the formation of a self-assembled monolayer to achieve the covalent binding of BMP-2 via its free amine residues. This method is a useful tool to study signaling at the cell membrane.

공유 결합은 자기 조립 단분자막의 접근 방식을 사용하여 표면에 BMP-2의 결합

Bone morphogenetic protein 2 (BMP-2) is a growth factor embedded in the extracellular matrix of bone tissue. BMP-2 acts as trigger of mesenchymal cell ...

Microwave-assisted Functionalization of Poly(ethylene glycol) and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large variety of PEG molecular weights and configurations (arm number, arm length, and branching pattern) affords researchers tight control over resulting hydrogel structures and properties, including Young&#8217;s modulus and mesh size. This video will illustrate a rapid, efficient, solvent-free, microwave-assisted method to methacrylate PEG precursors into poly(ethylene glycol) dimethacrylate (PEGDM). This synthetic method provides much-needed starting materials for applications in drug delivery and regenerative medicine. The demonstrated method is superior to traditional methacrylation methods as it is significantly faster and simpler, as well as more economical and environmentally friendly, using smaller amounts of reagents and solvents. We will also demonstrate an adaptation of this technique for on-resin methacrylamide functionalization of peptides. This on-resin method allows the N-terminus of peptides to be functionalized with methacrylamide groups prior to deprotection and cleavage from resin. This allows for selective addition of methacrylamide groups to the N-termini of the peptides while amino acids with reactive side groups (e.g.&#160;primary amine of lysine, primary alcohol of serine, secondary alcohols of threonine, and phenol of tyrosine) remain protected, preventing functionalization at multiple sites. This article will detail common analytical methods (proton Nuclear Magnetic Resonance spectroscopy (;H-NMR) and Matrix Assisted Laser Desorption Ionization Time of Flight mass spectrometry (MALDI-ToF)) to assess the efficiency of the functionalizations. Common pitfalls and suggested troubleshooting methods will be addressed, as will modifications of the technique which can be used to further tune macromer functionality and resulting hydrogel physical and chemical properties. Use of synthesized products for the formation of hydrogels for drug delivery and cell-material interaction studies will be demonstrated, with particular attention paid to modifying hydrogel composition to affect mesh size, controlling hydrogel stiffness and drug release.

Microwave-assisted Functionalization of Polyethylene glycol and On-resin Peptides for Use in Chain Polymerizations and Hydrogel Formation

This video will illustrate a rapid, efficient method to methacrylate poly(ethylene glycol), enabling chain polymerizations and hydrogel synthesis. It will demonstrate how to similarly introduce methacrylamide functionalities into peptides, detail common analytical methods to assess functionalization efficiency, provide suggestions for troubleshooting and advanced modifications, and demonstrate typical hydrogel characterization techniques.

전자 레인지를 이용한 폴리의 기능화 (에틸렌 글리콜) 및 체인 중합 및 하이드로 겔 형성에있는 사용을위한 온 수지 펩티드

One of the main benefits to using poly(ethylene glycol) (PEG) macromers in hydrogel formation is synthetic versatility. The ability to draw from a large ...

Formation of Ordered Biomolecular Structures by the Self-assembly of Short Peptides

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control self-assembly and mimicking this process in vitro will bring about major advances in the areas of materials science and nanotechnology. Among the available biological building blocks, peptides have several advantages as they present substantial diversity, their synthesis in large scale is straightforward, and they can easily be modified with biological and chemical entities1,2. Several classes of designed peptides such as cyclic peptides, amphiphile peptides and peptide-conjugates self-assemble into ordered structures in solution. Homoaromatic dipeptides, are a class of short self-assembled peptides that contain all the molecular information needed to form ordered structures such as nanotubes, spheres and fibrils3-8. A large variety of these peptides is commercially available.
This paper presents a procedure that leads to the formation of ordered structures by the self-assembly of homoaromatic peptides. The protocol requires only commercial reagents and basic laboratory equipment. In addition, the paper describes some of the methods available for the characterization of peptide-based assemblies. These methods include electron and atomic force microscopy and Fourier-Transform Infrared Spectroscopy (FT-IR). Moreover, the manuscript demonstrates the blending of peptides (coassembly) and the formation of a &#34;beads on a string&#34;-like structure by this process.9 The protocols presented here can be adapted to other classes of peptides or biological building blocks and can potentially lead to the discovery of new peptide-based structures and to better control of their assembly.

This paper describes the formation of highly ordered peptide-based structures by the spontaneous process of self-assembly. The method utilizes commercially available peptides and common lab equipment. This technique can be applied to a large variety of peptides and may lead to the discovery of new peptide-based assemblies.

짧은 펩타이드의 자기 조립에 의해 주문 생체 고분자 구조의 형성

In nature, complex functional structures are formed by the self-assembly of biomolecules under mild conditions. Understanding the forces that control ...

Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice through their ice-binding surfaces. Fluorescence-based ice plane affinity (FIPA) analysis is a modified technique used to determine the ice planes to which the AFPs bind. FIPA is based on the original ice-etching method for determining AFP-bound ice-planes. It produces clearer images in a shortened experimental time. In FIPA analysis, AFPs are fluorescently labeled with a chimeric tag or a covalent dye then slowly incorporated into a macroscopic single ice crystal, which has been preformed into a hemisphere and oriented to determine the a- and c-axes. The AFP-bound ice hemisphere is imaged under UV light to visualize AFP-bound planes using filters to block out nonspecific light. Fluorescent labeling of the AFPs allows real-time monitoring of AFP adsorption into ice. The labels have been found not to influence the planes to which AFPs bind. FIPA analysis also introduces the option to bind more than one differently tagged AFP on the same single ice crystal to help differentiate their binding planes. These applications of FIPA are helping to advance our understanding of how AFPs bind to ice to halt its growth and why many AFP-producing organisms express multiple AFP isoforms.

Antifreeze proteins (AFPs) bind to specific planes of ice to prevent or slow ice growth. Fluorescence-based ice plane affinity (FIPA) analysis is a modification of the original ice-etching method for determination of AFP-bound ice planes. AFPs are fluorescently labeled, incorporated into macroscopic single ice crystals, and visualized under UV light.

형광 기반 얼음 비행기 선호도에 의해 부동액 단백질의 얼음 결합 평면 결정

Antifreeze proteins (AFPs) are expressed in a variety of cold-hardy organisms to prevent or slow internal ice growth. AFPs bind to specific planes of ice ...

Water in Oil Emulsions: A New System for Assembling Water-soluble Chlorophyll-binding Proteins with Hydrophobic Pigments

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. Their functionality critically depends on their specific organization within large and elaborate multisubunit transmembrane protein complexes. In order to understand at the molecular level how these complexes facilitate solar energy conversion, it is essential to understand protein-pigment, and pigment-pigment interactions, and their effect on excited dynamics. One way of gaining such understanding is by constructing and studying complexes of Chls with simple water-soluble recombinant proteins. However, incorporating the lipophilic Chls and BChls into water-soluble proteins is difficult. Moreover, there is no general method, which could be used for assembly of water-soluble proteins with hydrophobic pigments. Here, we demonstrate a simple and high throughput system based on water-in-oil emulsions, which enables assembly of water-soluble proteins with hydrophobic Chls. The new method was validated by assembling recombinant versions of the water-soluble chlorophyll binding protein of Brassicaceae plants (WSCP) with Chl a. We demonstrate the successful assembly of Chl a using crude lysates of WSCP expressing E. coli cell, which may be used for developing a genetic screen system for novel water-soluble Chl-binding proteins, and for studies of Chl-protein interactions and assembly processes.

This manuscript describes a simple and high-throughput method for assembling water-soluble proteins with hydrophobic pigments that is based on water-in-oil emulsions. We demonstrate the effectiveness of the method in the assembly of native chlorophylls with four variants of recombinant water-soluble-chlorophyll binding proteins (WSCPs) of Brassica plants expressed in E. coli.

오일 유제에서 물 : 소수성 안료와 수용성 엽록소 결합 단백질을 조립하기위한 새로운 시스템

Chlorophylls (Chls) and bacteriochlorophylls (BChls) are the primary cofactors that carry out photosynthetic light harvesting and electron transport. ...

Radiolabeling and Quantification of Cellular Levels of Phosphoinositides by High Performance Liquid Chromatography-coupled Flow Scintillation

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular identity and function. Each of the seven PtdInsPs varies in their distribution and abundance, which are tightly regulated by specific kinases and phosphatases. The abundance of PtdInsPs can change abruptly in response to various signaling events or disturbance of the regulatory machinery. To understand how these events lead to changes in the amount of PtdInsPs and their resulting impact, it is important to quantify PtdInsP levels before and after a signaling event or between control and abnormal conditions. However, due to their low abundance and similarity, quantifying the relative amounts of each PtdInsP can be challenging. This article describes a method for quantifying PtdInsP levels by metabolically labeling cells with 3H-myo-inositol, which is incorporated into PtdInsPs. Phospholipids are then precipitated and deacylated. The resulting soluble 3H-glycero-inositides are further extracted, separated by high-performance liquid chromatography (HPLC), and detected by flow scintillation. The labeling and processing of yeast samples is described in detail, as well as the instrumental setup for the HPLC and flow scintillator. Despite losing structural information regarding acyl chain content, this method is sensitive and can be optimized to concurrently quantify all seven PtdInsPs in cells.

Phosphoinositides are signaling lipids whose relative abundance rapidly changes in response to various stimuli. This article describes a method to measure the abundance of phosphoinositides by metabolically labeling cells with 3H-myo-inositol, followed by extraction and deacylation. Extracted glycero-inositides are then separated by high-performance liquid chromatography and quantified by flow scintillation.

고성능 액체 크로마토 그래피 결합 흐름 섬광에 의한 방사성 표지 및 Phosphoinositides의 세포 수준의 정량화

Phosphoinositides (PtdInsPs) are essential signaling lipids responsible for recruiting specific effectors and conferring organelles with molecular ...

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum Interface (SALVI) and time-of-flight secondary ion mass spectrometry (ToF-SIMS). The fibronectin protein film was immobilized on the silicon nitride (SiN) membrane that forms the SALVI detection area. During ToF-SIMS analysis, three modes of analysis were conducted including high spatial resolution mass spectrometry, two-dimensional (2D) imaging, and depth profiling. Mass spectra were acquired in both positive and negative modes. Deionized water was also analyzed as a reference sample. Our results show that the fibronectin film in water has more distinct and stronger water cluster peaks compared to water alone. Characteristic peaks of amino acid fragments are also observable in the hydrated protein ToF-SIMS spectra. These results illustrate that protein molecule adsorption on a surface can be studied dynamically using SALVI and ToF-SIMS in the liquid environment for the first time.

In Situ Characterization of Hydrated Proteins in Water by SALVI and ToF-SIMS

This work presents a protocol for liquid handling and sample introduction to a microchannel for in situ time-of-flight secondary ion mass spectrometry analysis of protein biomolecules in an aqueous solution.

SALVI 및으로 ToF-SIMS에 의해 물에 수화 된 단백질의 제자리의 특성에

This work demonstrates in situ characterization of protein biomolecules in the aqueous solution using the System for Analysis at the Liquid Vacuum ...

DNA-magnetic Particle Binding Analysis by Dynamic and Electrophoretic Light Scattering

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the evaluation of DNA-magnetic particles binding via dynamic light scattering (DLS) and electrophoretic light scattering (ELS). Analysis by DLS provides valuable information on the physicochemical properties of particles including particle size, polydispersity, and zeta potential. The latter describes the surface charge of the particle which plays major role in electrostatic binding of materials such as DNA. Here, a comparative analysis exploits three chemical modifications of nanoparticles and microparticles and their effects on DNA binding and elution. Chemical modifications by branched polyethylenimine, tetraethyl orthosilicate and (3-aminopropyl)triethoxysilane are investigated. Since DNA exhibits a negative charge, it is expected that zeta potential of particle surface will decrease upon binding of DNA. Forming of clusters should also affect particle size. In order to investigate the efficiency of these particles in isolation and elution of DNA, the particles are mixed with DNA in low pH (~6), high ionic strength and dehydration environment. Particles are washed on magnet and then DNA is eluted by Tris-HCl buffer (pH = 8). DNA copy number is estimated using quantitative polymerase chain reaction (PCR). Zeta potential, particle size, polydispersity and quantitative PCR data are evaluated and compared. DLS is an insightful and supporting method of analysis that adds a new perspective to the process of screening of particles for DNA isolation.

This protocol describes the synthesis of magnetic particles and evaluation of their DNA-binding properties via dynamic and electrophoretic light scattering. This method focuses on monitoring changes in particle size, their polydispersity and zeta potential of particle surface which play major role in binding of materials such as DNA.

역동적이 고 전기 이동 산란 분석 바인딩 DNA-마그네틱 입자

Isolation of DNA using magnetic particles is a field of high importance in biotechnology and molecular biology research. This protocol describes the ...