PLD는 단백질 공학에서 캐나다 연구 위원장을 보유하고 있습니다. 이 작품은 PLD에 건강 연구의 캐나다 연구소에서 보조금에 의해 지원되었다. 이 연구는 또한 일본 과학진흥협회(JSPS)(제23310171호)와 일본 바이오 중심기술연구진흥기관(BRAIN)의 과학연구를 위한 보조금 지원에도 의해 지원되었습니다. 우리는 FIPA로 이어진 선구적인 작업에 대한 박사 크리스 마샬과 마이크 쿠이퍼에 감사드립니다. 우리는 또한 이 사업의 일부에 대한 시설을 제공하고 형광 광 여기 및 방출 필터를 설정한 로리 그레이엄 박사님께 도리어 사카에 츠다 박사님께 감사드립니다.

1. 성장하는 단일 얼음 결정
<ol><li>깨끗한 금속 팬 (직경 15cm, 높이 4.5cm)을 가지고 에틸렌 글리콜 냉각 욕조에 장착할 수 있습니다.</li>
	<li>파이프에서 단면을 톱질하여 폴리염화비닐(PVC) 원통형 몰드(직경 4.5cm, 높이 3-4cm, 두께 4mm)를 준비한다.<ol>
<li>한쪽에 노치(폭 1mm, 높이 2mm)를 자른다(그림 1A).</li>
		<li>팬에 편안하게 맞을 수 있는 금형을 최대한 준비합니다(그림 1B) 참고: 연구에 따르면 폴리비닐 알코올(PVA)이 얼음핵(22)에영향을 줄 수 있는 것으로 나타났습니다. 그러나 개방형 금형 PVC 시스템에서는 비정형 얼음 형성에 문제가 발생하지 않았습니다.</li>
	</ol>
</li>
	<li>노치 컷이 있는 표면인 각 금형의 바닥 표면 링에 진공 그리스의 가벼운 필름을 적용합니다. 노치가 팬 의 중심에서 멀리 방향을 그리는 금속 팬에 기름칠한 노치 표면을 밀봉합니다. 노치를 기름으로 채우거나 방해하지 않도록 주의하십시오.</li>
	<li>0.22 μm을 팬 중앙에 여과 및 탈기/탈기/탈온된 물을 추가하지만 금형 바깥쪽에 서서히 노치를 통해 금형에 물이 들어갈 수 있도록 합니다. 거품을 소개하지 않도록주의하십시오. 물 층은 약 5mm 깊이여야 합니다.</li>
	<li>팬을 온도 조절에 따라 -0.5°C로 냉각된 에틸렌 글리콜 목욕에 넣습니다. 팬은 완벽하게 평평해야합니다. 필요한 경우 팬 의 측면에 밸러스트 웨이트를 추가합니다.</li>
	<li>팬과 물이 -0.5 °C에 도달 한 후, 금형의 외부, 팬의 중간에 얼음의 작은 조각을 추가합니다.<ol>
<li>이것은 팬을 가로 질러 금형에 과냉각 된 물에 얼음 성장을 핵 것입니다. 각 금형의 하단에 있는 작은 노치를 통해 하나의 얼음 크리스탈만 전파할 수 있으며, 그 결과 각 금형에 단일 얼음 결정이 생성됩니다.</li>
		<li>하룻밤 동안 배양하여 얼음 층을 형성합니다.</li>
	</ol>
</li>
	<li>다음 3일 동안 하루에 한 번 각 금형에 4°C 의 탈가스/탈이온수 13ml를 추가하고, 첫날 -0.8°C를 각각 첨가한 후 에틸렌 글리콜 목욕의 온도를 1일째에 -1.1°C, 3일째에는 -1.5°C로 떨어뜨린다.<ol>
<li>하룻밤 사이에 그 온도에서 배양.</li>
		<li>4일째가 되면 금형은 얼음으로 완전히 채워져야 합니다.</li>
	</ol>
</li>
	<li>팬에서 금형을 당겨 얼음 결정을 금형밖으로 밀어 내고 계량 보트와 같은 깨끗한 표면에 보관하여 처리하기 전에 약 1 시간 동안 -20 ° C 냉동고에 보관하십시오.</li>
	<li>많은 작은 단일 얼음 결정을 준비하는 대신, 기사23에의해 설명 된 바와 같이 볼륨에 큰 단일 얼음 결정 여러 리터를 제조 할 수 있습니다. 대형 얼음 결정은 -15 ~ -20 °C 냉동고로 덮여 보관하는 경우 1 년 이상 보관 할 수 있습니다. 단일 결정의 부분은 필요에 따라 톱으로 얼음 블록에서 절단 될 수 있습니다.</li>
</ol>2. 얼음 결정의 특이점 과 방향을 결정
<ol><li>금형에서 배출된 얼음이 두 개의 교차 폴라로이드(그림 1D)사이에 있는 냉동실에서 관찰하여 단일 결정인지 확인합니다.<ol>
<li>얼음 결정이 단일인 경우 균열이나 불연속성을 볼 수 없으며 얼음 결정 내에서 빛의 방향이 바뀌어서는 안 됩니다. 참고: 냉동실을 사용할 수 없는 경우 필요한 모든 단계에서 콜드룸을 사용할 수 있으며, 신속하게 작업하고 얼음을 아껴서 처리해야 합니다.</li>
	</ol>
</li>
	<li>얼음 비경작으로 인해 c-축의 방향을 동시에 결정할 수 있습니다. 다음 정보를 사용하여 방향을 결정합니다.<ol>
<li>c-축이인시던트 라이트와 정확히 평행하면 이론적으로 교차된 폴라로이드를 통과하는 빛은 없습니다. 입사 라이트가 c-축과평행하여 약간 제목이 있는 경우 교차 폴라로이드 사이에서 회전할 때 균일한 다색 스펙트럼의 빛이 크리스탈을 통해 전달됩니다. 이 균일한 전송은 c-축24를따라 얼음 Ih의 광학 비활성에서 발생합니다. 얼음 결정의 기저 평면은 c-축에정상입니다.</li>
		<li>c-축이입사 광과 평행에서 교차 폴라로이드 사이에 회전하면, 전달된 빛은 크리스탈의 회전마다 0-100% 송전을 번갈아 가며 회전합니다.</li>
		<li>가장 일반적으로 c-축은원통형 얼음 결정의 원형 평면으로 정상이 될 것입니다.</li>
	</ol>
</li>
	<li>알루미늄 호일에 얼음 결정을 단단히 감싸고, 호일을 통해 바늘로 작은 구멍을 뚫고 기저 평면(c-축까지 정상)에 넣고 20분 동안 진공 아래에 놓음으로써 -축의방향을 결정합니다.<ol>
<li>이 치료는 기저 평면에 육각대 대칭으로 육각형 대칭을 가진 에칭을 생성합니다, 여기서 -축은6 면 별 (그림 2A)의정점을 통해 실행됩니다.</li>
		<li>원하는 경우, 얼음 결정은 각각 차가운 손가락에 수직으로 1차 또는 이차 프리즘 평면으로 장착하기 위해 육각형의 한쪽에 평행 또는 수직으로 톱으로 절단될 수 있다(그림 3).</li>
	</ol>
</li>
</ol>3. 단일 얼음 결정에 형광 라벨부동액 단백질의 흡착
<ol><li>먼저 크리스탈의 상단에 캐비티를 지루하게하여 차가운 손가락 (그림 1C)에 단일 얼음 결정을 마운트합니다. 이렇게하려면, 약간 다른 직경의 두 개의 알루미늄 막대로 얼음을 녹여, 그러나 차가운 손가락의 직경과 유사, 차가운 손가락이 맞을 수있는 구멍을 형성한다.</li>
	<li>차가운 손가락을 -0.5°C로 식히고 얼음 구멍에 놓고 얼음 결정을 금속으로 동결할 때까지 제자리에 고정시한다(도 2B). 손가락에서 막대로 의기온을 효율적으로 전달하는 것을 방해하기 때문에 손가락에 크리스탈을 부착할 때 기포를 피하십시오.</li>
	<li>얼음 결정의 직경의 약 2배에 달하는 반구형 컵을 여과된 탈온화 물 또는 완충액으로 채우고 약 4°C로 냉각시다. 차가운 손가락바인딩 얼음 결정을 컵에 담그고 얼음 결정의 상단이 액상층과 약 수준이며 얼음이 컵 벽을 만지지 않도록 과도한 물이나 버퍼를 제거합니다.<ol>
<li>단열재로 컵을 덮고 온도를 -5 °C로 낮춥춥습니다.</li>
		<li>얼음 결정이 반구로 형성될 때까지 약 1시간 정도 기다려 약 20분마다 상태를 확인합니다(그림 2C).</li>
		<li>얼음은 얼음 결정을 녹이고 성장시킴으로써 반구형 컵의 모양을 취할 것이지만, 컵 벽을 만지기 위해 결코 자라서는 안됩니다. 벽과 반구 사이에 는 적어도 1cm 간격이 있어야하며 차가운 손가락은 얼음에서 튀어 나가지 않아야합니다.</li>
	</ol>
</li>
	<li>얼음 결정에서 컵을 제거하고 25-30ml의 최종 부피와 원하는 분석 농도에 형광 단백질 용액을 추가하여 컵의 총 액체 부피를 변하지 않도록 주의하십시오. 전형적인 AFP 농도는 0.1 mg/ml입니다.<ol>
<li>얼음 결정의 상단이 액체와 수준에 있고 얼음 결정이 컵 벽을 만지지 않도록 컵에 얼음 결정을 다시 병합 (도 2D).</li>
		<li>차가운 손가락 온도를 -8°C로 떨어뜨리고 단백질 용액이 얼음 결정으로 2-3 시간 동안 동결되어 용액을 자주 저어줍니다. 단백질 용액에서 형성된 얼음은 얼음 성장을 멈추기 전에 적어도 5mm여야 합니다.</li>
	</ol>
</li>
	<li>차가운 손가락에 부착된 상태에서 컵에서 얼음 결정을 제거합니다. 차가운 손가락을 통해 냉각수를 0°C 이상으로 데우고 얼음 결정이 녹을 때까지 기다립니다.</li>
	<li>결정평면면을 계량 접시와 같은 깨끗한 표면에 내려놓고 새로 형성된 얼음을 만지지 않도록 주의하고 -20°C에서 최소 20분 동안 보관하십시오.</li>
</ol>4. 부동액 단백질 바인딩 얼음 비행기의 시각화
<ol><li>형광의 시각화는 어두운 냉동고 또는 콜드 룸에서 수행되며, 파장 특정 흥분 필터가있는 램프 아래에 얼음 결정 평면 면을 배치하여 다른 비특이적 인 빛을 차단합니다. 패턴에 따라, AfP에 의해 바인딩되는 얼음 비행기를 추정 할 수있다 (그림 4).</li>
	<li>파장 특정 조명을 사용할 수 없는 경우 UV 라이트 박스를 대신 사용할 수 있습니다.</li>
	<li>전통적인 얼음 등광은 얼음 반구가 적어도 3 시간 동안 -20 °C에서 승화할 수 있도록 함으로써 간단하게 수행 될 수 있으며, 그 후에 잔류 단백질 분말이 얼음 표면 (그림 5)에서볼 수 있습니다.</li>
	<li>2.3단계는 완성된 얼음 반구의 -축의방향을 결정하기 위해 반복될 수 있다.</li>
</ol>

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P., 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=Beta-helix+structure+and+ice-binding+properties+of+a+hyperactive+antifreeze+protein+from+an+insect.">Beta-helix structure and ice-binding properties of a hyperactive antifreeze protein from an insect.</a> Nature. 406 (6793), 325-328 (2000).</li><li>Pertaya, N., Marshall, C. B., Celik, Y., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+visualization+of+spruce+budworm+antifreeze+protein+interacting+with+ice+crystals:+Basal+plane+affinity+confers+hyperactivity.">Direct visualization of spruce budworm antifreeze protein interacting with ice crystals: Basal plane affinity confers hyperactivity.</a> Biophys. J. 95 (1), 333-341 (2008).</li><li>Middleton, A. J., 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=Antifreeze+protein+from+freeze-tolerant+grass+has+a+beta-roll+fold+with+an+irregularly+structured+ice-binding+site.">Antifreeze protein from freeze-tolerant grass has a beta-roll fold with an irregularly structured ice-binding site.</a> J. Mol. Biol. 416 (5), 713-724 (2012).</li><li>Scotter, A. J., 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+basis+for+hyperactivity+of+antifreeze+proteins.">The basis for hyperactivity of antifreeze proteins.</a> Cryobiology. 53 (2), 229-239 (2006).</li><li>Knight, C. A., Wierzbicki, A., Laursen, R. A., 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=Adsorption+of+biomolecules+to+ice+and+their+effects+upon+ice+growth.+1.+Measuring+adsorption+orientations+and+initial+results.">Adsorption of biomolecules to ice and their effects upon ice growth. 1. Measuring adsorption orientations and initial results.</a> Crys. Growth Des. 1 (6), 429-438 (2001).</li><li>Hakim, A., 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=Crystal+structure+of+an+insect+antifreeze+protein+and+its+implications+for+ice+binding.">Crystal structure of an insect antifreeze protein and its implications for ice binding.</a> J. Biol. Chem. 288 (17), 12295-12304 (2013).</li><li>Garnham, C. P., Nishimiya, Y., Tsuda, S., Davies, P. 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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=A+simple+technique+for+growing+large,+optically+''perfect''+ice+crystals.">A simple technique for growing large, optically ''perfect'' ice crystals.</a> J. Glac. 42 (142), 585-587 (1996).</li><li>Hobbs, P. 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> Ice physics. , 200-248 (1974).</li><li>Knight, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Formation+of+crystallographic+etch+pits+on+ice,+and+its+application+to+the+study+of+hailstones.">Formation of crystallographic etch pits on ice, and its application to the study of hailstones.</a> J. Appl. Meteorol. 5 (5), 710-714 (1966).</li><li>Yang, D. S., Sax, M., Chakrabartty, A., Hew, C. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Crystal+structure+of+an+antifreeze+polypeptide+and+its+mechanistic+implications.">Crystal structure of an antifreeze polypeptide and its mechanistic implications.</a> Nature. 333 (6170), 232-237 (1988).</li><li>Sicheri, F., Yang, D. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ice-binding+structure+and+mechanism+of+an+antifreeze+protein+from+winter+flounder.">Ice-binding structure and mechanism of an antifreeze protein from winter flounder.</a> Nature. 375 (6530), 427-431 (1995).</li><li>Devries, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+glycopeptides+and+peptides+in+inhibition+of+crystallization+of+water+in+polar+fishes.">Role of glycopeptides and peptides in inhibition of crystallization of water in polar fishes.</a> Philos. T Roy. Soc. B. 304 (1121), 575-588 (1984).</li><li>Pertaya, N., 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=Fluorescence+microscopy+evidence+for+quasi-permanent+attachment+of+antifreeze+proteins+to+ice+surfaces.">Fluorescence microscopy evidence for quasi-permanent attachment of antifreeze proteins to ice surfaces.</a> Biophys. J. 92 (10), 3663-3673 (2007).</li><li>Kondo, H., 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=Ice-binding+site+of+snow+mold+fungus+antifreeze+protein+deviates+from+structural+regularity+and+high+conservation.">Ice-binding site of snow mold fungus antifreeze protein deviates from structural regularity and high conservation.</a> Proc. Natl. Acad. Sci. U.S.A. 109 (24), 9360-9365 (2012).</li><li>Mok, Y. F., 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=Structural+basis+for+the+superior+activity+of+the+large+isoform+of+snow+flea+antifreeze+protein.">Structural basis for the superior activity of the large isoform of snow flea antifreeze protein.</a> Biochemistry. 49 (11), 2593-2603 (2010).</li><li>Takamichi, M., Nishimiya, Y., Miura, A., Tsuda, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fully+active+qae+isoform+confers+thermal+hysteresis+activity+on+a+defective+sp+isoform+of+type+iii+antifreeze+protein.">Fully active qae isoform confers thermal hysteresis activity on a defective sp isoform of type iii antifreeze protein.</a> FEBS J. 276 (5), 1471-1479 (2009).</li><li>Bar-Dolev, M., Celik, Y., Wettlaufer, J. S., Davies, P. L., Braslavsky, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+ice+growth+and+melting+modifications+by+antifreeze+proteins.">New insights into ice growth and melting modifications by antifreeze proteins.</a> J. R. Soc. Interface. 9 (77), 3249-3259 (2012).</li><li>Celik, 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=Microfluidic+experiments+reveal+that+antifreeze+proteins+bound+to+ice+crystals+suffice+to+prevent+their+growth.">Microfluidic experiments reveal that antifreeze proteins bound to ice crystals suffice to prevent their growth.</a> Proc. Natl. Acad. Sci. U.S.A. 110 (4), 1309-1314 (2013).</li><li>Garnham, C. P., Campbell, R. L., Walker, V. K., Davies, P. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+dimeric+beta-helical+model+of+an+ice+nucleation+protein+with+bridged+active+sites.">Novel dimeric beta-helical model of an ice nucleation protein with bridged active sites.</a> BMC Struct. Biol. 11, (2011).</li></ol>

이해 상충이 선언되지 않았습니다.

AFP 바인딩 아이스 비행기의 결정에 대한 찰스 나이트에 의해 얼음 에칭 방법의 개발은 크게 AFP에 의해 얼음 바인딩의 메커니즘에 대한 연구를 고급. Afp의 구조는 X 선 결정학26,27에 의해 해결 될 수있는 반면 AFP가 결합하는 얼음에 보완 표면을 추론하는 명백한 방법은 없었다. 겨울 가자미에서 I 타입 I AFP가 처음에 특징지어졌을 때, 얼음28의주요 프리즘 비행기에 결합하는 것이 가?...

부동액 단백질 (AfP)의 생산은 얼음이 가득한 환경에 사는 일부 유기체의 중요한 생존 메커니즘입니다. 최근까지, AFP의 유일한 기능은 혈액 순환을 막고 조직 손상을 일으키고 삼투압을 유발하는 내부 얼음 결정의 성장을 방지하거나 늦추는 것이라고 생각되었습니다. 물고기와 같은 동결정도를 견딜 수 없는 유기체는 얼음 결정 성장을 완전히 저해하기 위해 AFP를발현한다 1. 잔디와 같은 다른 것들은 동결 관용이며 조직에 큰 얼음 결정의 형성을 감소시키는 얼음 재결정을 억제하기 위해 AFP를 발현한다2. 저온에서 멤브레인의 안정화는 AFP3에대해 제안된 또 다른 기능이다. 최근, 얼음으로 덮인 황천 호수4에서남극 박테리아 마리노모나스 원시인의AFP에 대한 새로운 역할이 제안되었습니다. 이 AFP는 훨씬 더 큰 접착 단백질의일부입니다 5 산소와 영양소에 더 나은 액세스를 위해 얼음에 박테리아를 부착 하는 것으로 생각 되는6. 그밖 미생물은7에사는 얼음의 구조물을 바꿀 수 있는 AFP를 분비하기 위하여 알려지고 있습니다.
일부 물고기, 곤충, 식물, 조류, 박테리아, 규격 및 곰팡이에서 Afp가 발견되었습니다. 그들은 여러 경우에 다른 선조에서 그들의 진화와 일치 하는 현저하게 발산 순서와 구조; 그러나 그들은 모두 얼음에 결합하고 흡착 억제 메커니즘 8에 의해 성장을억제한다. AFP에는 각각 얼음 바인딩 사이트(IBS)의 역할을 하는 특정 표면이 있습니다. 이들은 전형적으로 표면 잔류물의 사이트 지시 돌연변이 발생에 의해 확인되었습니다9-11. IBS는 얼음의 특정 평면과 일치하는 얼음 모양의 패턴으로 물 분자를 배열하는 가설입니다. 따라서 AFP는5, 12에바인딩하기 전에 리간드를 형성한다. 얼음 비행기는 밀러 지수에 의해 정의될 수 있으며, 다른 AFP는 다른 평면에 묶을 수 있습니다. 따라서, 겨울 가마에서 타입 I AFP는 20-21 피라미드 평면(13)에바인딩, 유형 III AFP는 복합 얼음 바인딩 표면을 사용하여 기본 프리즘과 피라미드 평면을 모두 결합11,14,가문비 나무 budworm AFP, 활동적인 AFP, 기본 및 기저 평면 모두에 동시에 결합15,16. MpAFP와 같은 다른 활동적인 AFP는 단일 얼음 결정 반구5,17의완전한 커버리지에 의해 표시된 바와 같이 여러 얼음 비행기에 결합합니다. 기저 평면뿐만 아니라 다른 평면을 묶는 활동적인 AFP의 능력이 적당히 활성18을통해 10 배 더 높은 활동을 차지할 수 있다고 가설이 있습니다. 활동적인 AF의 효율성은 잘 문서화되어 있지만 여러 얼음 비행기에 결합하는 능력은 아직 이해되지 않습니다.
AFP 바인딩 얼음 비행기를 결정하는 원래 방법은 찰스 나이트에 의해 개발되었다13,19. 이 방법에서 거시적인 단일 얼음 결정은 중공 금속 막대 (차가운 손가락)에 장착되어 탈가스물로 채워진 반구 컵에 침수하여 반구로 형성됩니다. 이어서, 반구는 AFP의 희석용액으로 침수되고, 차가운 손가락을 통해 순환하는 에틸렌 글리콜의 온도에 의해 제어되는 몇 시간 동안 AFP 용액에서 얼음 결정 반구로 얼음층이 재배된다. 얼음 결정은 용액에서 제거되고, 차가운 손가락에서 분리하고- 10 ~ -15 °C 냉동실에 놓습니다. 표면은 부동액 단백질 용액의 냉동 표면 필름을 제거하기 위해 날카로운 블레이드로 긁어 내고 얼음 결정은 적어도 3 시간 동안 승화 할 수 있습니다. 승화 후, AfP에 의해 결합된 얼음 비행기는 잔류 단백질에서 파생된 백색 에칭 패턴으로 볼 수 있다. 얼음 반구는 c-축및-축을 향하여 얼음의 기저 및 프리즘 평면을 찾고 에칭 패치의 밀러 지수를 결정할 수 있습니다. 
여기서우리는 형광기반 얼음 평면 친화성(FIPA)11로지칭하는 방법인 얼음의 AFP 바운드 평면을 결정하는 본래 방법의 수정을 설명한다. AfPs는 녹색 형광 단백질 (GFP)11,16,17,20과같은 키메라 태그 또는 AFP5,21에공유결합된 형광 염료로 형광으로 표시된다. 형광으로 표시된 AfP는 단일 얼음 결정으로 흡착되고 원래얼음 에칭 실험과 동일한 실험 절차를 사용하여 자란다. 성장하는 얼음 반구에 결합하는 AFP의 범위는 자외선(UV) 램프를 사용하여 실험 전반에 걸쳐 모니터링될 수 있다. 실험이 완료되면 반구는 차가운 손가락에서 직접 분리하고 승화없이 이미지화 될 수 있습니다. 그러나 원하는 경우 반구를 상화하여 기존의 얼음 식각을 시각화할 수 있습니다. FIPA 방법론에 도입된 수정은 기존의 얼음 에칭 프로토콜을 몇 시간 단축시합니다. 또한, AFP 바인딩 된 얼음 비행기의 중복 패턴을 시각화하기 위해 서로 다른 형광 라벨을 가진 여러 AfP를 동시에 이미징 할 가능성이 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="605">
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	</colgroup>
	<tbody>
		<tr height="42">
			<td height="42" style="height:42px;width:177px;">NESLAB RTE Refrigerating Bath/Circulators</td>
			<td style="width:95px;">Thermo Scientific</td>
			<td style="width:145px;">RTE7</td>
			<td style="width:189px;"></td>
		</tr>
		<tr height="48">
			<td height="48" style="height:48px;width:177px;">Ethylene glycol, Premixed Antifreeze/Coolant</td>
			<td style="width:95px;">Certified</td>
			<td style="width:145px;">29-3037-0</td>
			<td style="width:189px;">Common automotive antifreeze</td>
		</tr>
		<tr height="65">
			<td height="65" style="height:65px;width:177px;">Cold finger</td>
			<td style="width:95px;">not available</td>
			<td style="width:145px;">not available</td>
			<td style="width:189px;">Custom made with brass (9 cm long, 1.5 cm outer diameter)</td>
		</tr>
		<tr height="67">
			<td height="67" style="height:67px;width:177px;">Hemispherical cup</td>
			<td style="width:95px;">not available</td>
			<td style="width:145px;">not available</td>
			<td style="width:189px;">Custom made with resin (8 cm outer diameter, 6 cm inner diameter)</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;width:177px;">High Dual Output Lighting System</td>
			<td style="width:95px;">Lightools Research</td>
			<td style="width:145px;">LT-99D2, Illumatools DLS 120 volts AC, LT-9470FX, LT-9549FX</td>
			<td style="width:189px;">Additional and custom excitation filters can be purchased from Lightools Research</td>
		</tr>
		<tr height="20">
			<td height="40" rowspan="2" style="height:40px;width:177px;">Camera</td>
			<td rowspan="2" style="width:95px;">Canon</td>
			<td rowspan="2" style="width:145px;">EOS 50D</td>
			<td rowspan="2" style="width:189px;"></td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="79">
			<td height="79" style="height:79px;width:177px;">Emission Filters</td>
			<td style="width:95px;">Lightools Research</td>
			<td style="width:145px;">LT-9EFPVG, LT-9GFPVG, LT-9RFPVG</td>
			<td style="width:189px;">Filter ring adapter may be required to fit filter onto camera lens</td>
		</tr>
	</tbody>
</table>

determining the ice-binding planes of antifreeze proteins by fluorescence-based ice plane affinity

부동액 단백질(Afp)은 내부 얼음 성장을 방지하거나 늦추기 위해 다양한 냉강한 유기체로 표현됩니다. AFP는 얼음 바인딩 표면을 통해 특정 얼음 평면에 바인딩합니다. 형광 계 얼음 평면 선호도 (FIPA) 분석은 AFP가 결합하는 얼음 비행기를 결정하는 데 사용되는 수정 된 기술입니다. FIPA는 AFP 바인딩 된 얼음 비행기를 결정하기위한 원래의 얼음 에칭 방법을 기반으로합니다. 실험 시간이 단축된 후 선명한 이미지를 생성합니다. FIPA 분석에서 AFP는 키메라 태그 또는 공유 염료로 형광으로 형광으로 표시된 다음 천천히 거시적 단일 얼음 결정으로 통합되어 반구로 미리 형성되어 a-및 c-축을결정합니다. AFP 바인딩 된 얼음 반구는 비특이적 인 빛을 차단하기 위해 필터를 사용하여 AFP 바인딩 평면을 시각화하기 위해 UV 빛 아래 이미지입니다. AFP의 형광 라벨링을 통해 AFP흡착을 실시간으로 모니터링할 수 있습니다. 레이블은 AF가 바인딩하는 비행기에 영향을 미치지 않는 것으로 나타났습니다. FIPA 분석은 또한 결합 평면을 차별화하는 데 도움이 동일한 단일 얼음 결정에 서로 다른 태그가 달린 AFP를 바인딩하는 옵션을 소개합니다. FIPA의 이러한 응용 프로그램은 AFP가 성장을 중지얼음에 바인딩하는 방법과 많은 AFP 생산 유기체가 여러 AFP 동위 형태를 표현하는 이유에 대한 우리의 이해를 발전시키는 데 도움이됩니다.

단일 얼음 결정의 준비 및 장착은 오류가 가장 일반적으로 이루어지는 FIPA 절차의 두 단계입니다. 준비된 얼음 결정이 단일인지 여부를 결정하는 것은 프로토콜 섹션의 2.1 단계에서 설명된 대로 교차 폴라로이드(도 1D)를통해 이를 검사하여 수행된다. FIPA 분석에 다결정 얼음 결정이 사용되는 경우, 그 결과는 일관된 결합 패턴(도 6B)없이반구에 대한 AFP의 불연속 결합이 될...

Watch this Scientific Journal Video about Determining the Ice-binding Planes of Antifreeze Proteins by Fluorescence-based Ice Plane Affinity at JoVE.com

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

부동액 단백질(AfP)은 얼음 의 특정 평면에 결합하여 얼음 성장을 방지하거나 느리게 합니다. 형광계 얼음 비행기 선호도(FIPA) 분석은 AFP 바인딩 된 얼음 비행기의 측정을위한 원래 얼음 에칭 방법을 수정한 것입니다. AFP는 형광으로 표시되고, 거시적 단일 얼음 결정에 통합되고, UV 빛 아래에서 시각화됩니다.

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 ...

determining-ice-binding-planes-antifreeze-proteins-fluorescence-based

Research

JoVE Journal

Chemistry

8.9K 조회수.  Queen's University. 부동액 단백질(AfP)은 얼음 의 특정 평면에 결합하여 얼음 성장을 방지하거나 느리게 합니다. 형광계 얼음 비행기 선호도(FIPA) 분석은 AFP 바인딩 된 얼음 비행기의 측정을위한 원래 얼음 에칭 방법을 수정한 것입니다. AFP는 형광으로 표시되고, 거시적 단일 얼음 결정에 통합되고, UV 빛 아래에서 시각화됩니다.

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

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

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

의 NMR 공명 확인하는 방법<sup&gt; 13</sup환원 메틸을 단백질에&gt; C-디메틸 N-말단 아민

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

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 ...

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the time-profile volume and enthalpy changes of light induced conformational changes in proteins on microsecond to millisecond time-scales that are not accessible using traditional stop-flow instruments. In addition, since overall changes in volume and/or enthalpy are probed, these techniques can be applied to proteins and other biomacromolecules that lack a fluorophore and or a chromophore label. To monitor dynamics and energetics of structural changes associated with Ca2+ binding to calcium transducers, such neuronal calcium sensors, a caged calcium compound, DM-nitrophen, is employed to photo-trigger a fast (&#964; &#60; 20 &#956;sec) increase in free calcium concentration and the associated volume and enthalpy changes are probed using photothermal beam deflection technique.

Here we report an application of the photothermal beam deflection technique in combination with a caged calcium compound, DM-nitrophen, to monitor microsecond and millisecond dynamics and energetics of structural changes associated with the calcium association to a neuronal calcium sensor, Downstream Regulatory Element Antagonist Modulator.

광열 편향 빔에 의해 해결 단백질의 밀리 초 이하의 구조적 변화

Photothermal beam deflection together with photo-acoustic calorimetry and thermal grating belongs to the family of photothermal methods that monitor the ...

Fluorescence-based Monitoring of PAD4 Activity via a Pro-fluorescence Substrate Analog

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein substrates. The histone tails represent one of the most heavily modified stretches within all human proteins. Peptidyl-arginine deiminase 4 (PAD4) has been shown to convert arginine residues into the non-genetically encoded citrulline residue. Few assays described to date have been operationally facile with satisfactory sensitivity. Thus, the lack of adequate assays has likely contributed to the absence of potent non-covalent PAD4 inhibitors. Herein a novel fluorescence-based assay that allows for the monitoring of PAD4 activity is described. A pro-fluorescent substrate analog was designed to link PAD4 enzymatic activity to fluorescence liberation upon the addition of the protease trypsin. It was shown that the assay is compatible with high-throughput screening conditions and has a strong signal-to-noise ratio. Furthermore, the assay can also be performed with crude cell lysates containing over-expressed PAD4.

PAD4 is an enzyme responsible for the conversion of peptidyl-arginine to peptidyl-citrulline. Dysregulation of PAD4 has been implicated in a number of human diseases. A facile and high-throughput compatible fluorescence based PAD4 assay is described.

프로 형광 기판 아날로그를 통해 PAD4 활동의 형광 기반의 모니터링

Post-translational modifications may lead to altered protein functional states by increasing the covalent variations on the side chains of many protein ...

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional units (e.g., domains). Traditional consensus-building models fail to account for interpositional dependencies &#8211; functionally required covariation of residues that tend to appear simultaneously throughout evolution and across the phylogentic tree. These relationships can reveal important clues about the processes of protein folding, thermostability, and the formation of functional sites, which in turn can be used to inform the engineering of synthetic proteins. Unfortunately, these relationships essentially form sub-motifs which cannot be predicted by simple &#8220;majority rule&#8221; or even HMM-based consensus models, and the result can be a biologically invalid &#8220;consensus&#8221; which is not only never seen in nature but is less viable than any extant protein. We have developed a visual analytics tool, StickWRLD, which creates an interactive 3D representation of a protein alignment and clearly displays covarying residues. The user has the ability to pan and zoom, as well as dynamically change the statistical threshold underlying the identification of covariants. StickWRLD has previously been successfully used to identify functionally-required covarying residues in proteins such as Adenylate Kinase and in DNA sequences such as endonuclease target sites.

Synthetic protein sequences based on consensus motifs typically ignore co-evolving residues, that imply interpositional dependencies (IPDs). IPDs can be essential to activity, and designs that disregard them may result in suboptimal results. This protocol uses StickWRLD to identify IPDs and help inform rational protein design, resulting in more efficient results.

합성 단백질의 최적화 : Interpositional 종속 관계 확인이 구조적으로 표시 및 / 또는 기능적으로 연결된 잔류

Protein alignments are commonly used to evaluate the similarity of protein residues, and the derived consensus sequence used for identifying functional ...

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 ...

Facile Synthesis of Worm-like Micelles by Visible Light Mediated Dispersion Polymerization Using Photoredox Catalyst

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins with the synthesis of a hydrophilic poly(oligo(ethylene glycol) methyl ether methacrylate) (POEGMA) homopolymer using reversible addition-fragmentation chain-transfer (RAFT) polymerization. Under mild visible light irradiation (&#955; = 460 nm, 0.7 mW/cm2), this macro-chain transfer agent (macro-CTA) in the presence of a ruthenium based photoredox catalyst, Ru(bpy)3Cl2 can be chain extended with a second monomer to form a well-defined block copolymer in a process known as Photoinduced Electron Transfer RAFT (PET-RAFT). When PET-RAFT is used to chain extend POEGMA with benzyl methacrylate (BzMA) in ethanol (EtOH), polymeric nanoparticles with different morphologies are formed in situ according to a polymerization-induced self-assembly (PISA) mechanism. Self-assembly into nanoparticles presenting POEGMA chains at the corona and poly(benzyl methacrylate) (PBzMA) chains in the core occurs in situ due to the growing insolubility of the PBzMA block in ethanol. Interestingly, the formation of highly pure worm-like micelles can be readily monitored by observing the onset of a highly viscous gel in situ due to nanoparticle entanglements occurring during the polymerization. This process thereby allows for a more reproducible synthesis of worm-like micelles simply by monitoring the solution viscosity during the course of the polymerization. In addition, the light stimulus can be intermittently applied in an ON/OFF manner demonstrating temporal control over the nanoparticle morphology.

This article describes a process for producing polymeric self-assembled nanoparticles using visible light mediated dispersion polymerization. Using low energy visible light to control the polymerization allows for the reproducible formation of self-assembled worm-like micelles at high solids content.

Photoredox 촉매를 사용하여 가시 광선 중재 분산 중합에 의해 웜 형상 미셀 손쉬운 합성

Presented herein is a protocol for the facile synthesis of worm-like micelles by visible light mediated dispersion polymerization. This approach begins ...

Determining the Chemical Composition of Corrosion Inhibitor/Metal Interfaces with XPS: Minimizing Post Immersion Oxidation

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, the focus is on metallic substrates immersed in acidic solutions containing organic corrosion inhibitors, as these systems can be particularly sensitive to oxidation following removal from solution. To minimize the likelihood of such degradation, samples are removed from solution within a glove box purged with inert gas, either N2 or Ar. The glove box is directly attached to the load-lock of the ultra-high vacuum X-ray photoelectron spectroscopy instrument, avoiding any exposure to the ambient laboratory atmosphere, and thus reducing the possibility of post immersion substrate oxidation. On this basis, one can be more certain that the X-ray photoelectron spectroscopy features observed are likely to be representative of the in situ submerged scenario, e.g. the oxidation state of the metal is not modified.

A protocol to avoid the oxidation of metallic substrates during sample transfer from an inhibited acidic solution to an X-ray photoelectron spectrometer is presented.

XPS와 부식 억제제 / 금속 인터페이스의 화학 성분을 결정 : 최소화 포스트 집중 산화

An approach for acquiring more reliable X-ray photoelectron spectroscopy data from corrosion inhibitor/metal interfaces is described. More specifically, ...

An HS-MRM Assay for the Quantification of Host-cell Proteins in Protein Biopharmaceuticals by Liquid Chromatography Ion Mobility QTOF Mass Spectrometry

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring high sensitivity and a wide dynamic range. Mass spectrometry-based quantification assays for proteins typically involve protein digestion followed by the selective reaction monitoring/multiple reaction monitoring (SRM/MRM) quantification of peptides using a low-resolution (Rs ~1,000) tandem quadrupole mass spectrometer. One of the limitations of this approach is the interference phenomenon observed when the peptide of interest has the "same" precursor and fragment mass (in terms of m/z values) as other co-eluting peptides present in the sample (within a 1-Da window). To avoid this phenomenon, we propose an alternative mass spectrometric approach, a high selectivity (HS) MRM assay that combines the ion mobility separation of peptide precursors with the high-resolution (Rs ~30,000) MS detection of peptide fragments. We explored the capabilities of this approach to quantify low-abundance peptide standards spiked in a monoclonal antibody (mAb) digest and demonstrated that it has the sensitivity and dynamic range (at least 3 orders of magnitude) typically achieved in HCP analysis. All six peptide standards were detected at concentrations as low as 0.1 nM (1 femtomole loaded on a 2.1-mm ID chromatographic column) in the presence of a high-abundance peptide background (2 &#181;g of a mAb digest loaded on-column). When considering the MW of rabbit phosphorylase (97.2 kDa), from which the spiked peptides were derived, the LOQ of this assay is lower than 50 ppm. Relative standard deviations (RSD) of peak areas (n = 4 replicates) were less than 15% across the entire concentration range investigated (0.1-100 nM or 1-1,000 ppm) in this study.

Here, we describe a chromatographic assay coupled with the ion mobility separation of peptide precursors followed by the high-resolution (~30,000) MS-detection of peptide fragments for the quantification of spiked peptide standards in a monoclonal antibody digest.

액체 크로마토그래피 이온 이동성 QTOF 질량 분석에 의해 단백질 바이오에 주인 세포 단백질의 정량화에 대 한 HS MRM 분석 결과

The analysis of low-level (1-100 ppm) protein impurities (e.g., host-cell proteins (HCPs)) in protein biotherapeutics is a challenging assay requiring ...