이 작품은 국립 과학 재단 (MCB 1,021,831, JM)와 J. &amp; E. 바이오 메디컬 연구 프로그램 (건강의 플로리다학과, JM)에 의해 지원되었다.

1. 샘플 준비 <ol><li> 샘플 준비하고 원하지 않는 uncaging을 방지하기 위해 어두운 방에있는 모든 샘플 조작을 수행하십시오. </li><li> 50 mM의 HEPES에 N, N, N &#39;, N&#39;- 테트라 키스 [(옥시 카르 보닐) 메틸] -1,2 - 에탄 디아민) - (2 - 니트로 -4,5 - 디메 톡시 페닐) - DM-nitrophen ((1 가용화 버퍼, 100 ㎜의 KCl, 400 μM의 최종 농도로 pH를 7.0 (= 350 ㎚ ε 4,330 M -1 cm -1 9). </li><li> [DM-nitrophen] : [칼슘 2 +]의 바람직한 비율을 달성하기 위해 0.1 M 주식 솔루션에서 염화칼슘을 추가합니다. 10 μM보다 큰 칼슘 2 + 협회 K d의 단백질, [칼슘 2 +]의 비율 : 1:1 [DM-nitrophen는] uncaged DM-nitrophen에 사진 출시 칼슘 2 +의 바인딩을 방지하는 것이 바람직하다 . 사실, 칼슘의 90 %는 결합 단백질 ~ 10 ㎚ 및 400 ㎜로 DM-nitrophen 및 CA 2 +의 총 농도로 DM-nitrophen의 K의 D 값을 고려K d (으)로 = 10 μM은 apoform에있을 것입니다. 한편, K의 D &lt;10 μM과 단백질에 결합하는 칼슘 2 +의 연구를 위해, 그것은 [칼슘 2 +] 감소하는 것이 바람직의 :과 칼슘 2 + 착물을 방지하기 위해 0.95 [DM-nitrophen] 비를 APO- 단백질 케이지 사진 해리 사전. </li><li> 기준 화합물 K 3 가용화 [철 (III) (CN)를 6] 또는 샘플과 동일한 버퍼 나 2 CRO 4. </li></ol> 2. 실험 설정하기 <ol><li> 기본적인 실험 구성은도 2에 도시된다. </li><li> 1mm로 프로브 빔의 직경 (HE-Ne 레이저의 632 nm의 출력, ~ 5 mW의 레이저 파워)를 조정하고 온도에 배치 셀의 중심을 통해 프로브 빔을 전파하는 핀홀 (P 2)를 사용 M 1 거울을 사용하여 셀 홀더를 통제했다. </li><li> 프로브 빔의 중심을 샘플 뒤에 거울 (M 2)를 사용하여위치에 민감한 검출기의 중심에. </li><li> 베스트 개의 다이오드와 바닥이 다이오드뿐만 아니라 감지기의 좌측 및 우측에서 두 개의 다이오드 사이의 전압 차 사이의 전압의 차이가 있음을 이러한 방식으로, 검출기의 중심에서 프로브 빔을 초점 제로. </li><li> YAG 레이저, FWHM 5 NSEC)이 355 nm의 레이저 거울 사이에 3mm의 핀홀 (P 1)를 사용 :이어서, 펌프 빔의 직경, Q-스위치의 Nd 355 nm의 출력을 형성. </li><li> 도 2에서와 같이 큐벳의 중심을 통해 펌프 빔 Copropagate. 그것은 두 개의 레이저 빔이 측정 가능한 편향 각 및 PBD 신호 따라서 높은 진폭을 구하는 것이 거의 동일 직선 방식으로 광학 셀의 중심을 통해 전파되는 것이 중요하다. 실험 조건 하에서, 프로브 및 펌프 빔의 교차 각도가 15 ° 미만이다. </li><li> 프로브 및 펌프를 정렬 기준 화합물을 사용하여더 긴 시간 척도 (~ 100 밀리 초)에서 양호한 S / N 비와 안정 PBD 진폭, 즉 만족 PBD 신호를 달성하기 위해 빔. </li><li> 355 nm의 레이저 미러의 증분 조정하여 프로브 빔에 대하여 펌프 빔의 위치를​​ 조정한다. </li><li> 위치 검출기에 민감한이 상하 포토 다이오드 사이의 차이로서 PBD 기준 신호의 진폭을 측정한다. PBD 신호는 빠른 시간 규모 (&lt;10 마이크로 초)에 진폭의 급격한 증가를 나타내고 그림 3에서와 같이 100 밀리 초 척도에서 안정적으로 유지해야합니다. PDB 진폭의 총 변동성에 슛은 신호 진폭의 5 % 내에서하고 신호 재현성는 주로 진동에 의해 영향을받습니다. </li><li> 흡수 된 여기 레이저 파워 온 아인슈타인의 개수에 PBD 신호의 선형 의존성, E의 측정에 의해 방출 열 에너지에 대하여 PBD 신호 진폭의 선형성을 확인= A는 여기 파장에서의 흡광도 기준에 대응한다 (1-10-A). </li><li> 레이저 아래 전력 약 1,000 μJ 및 다 광자 흡수와 각각 펌프 빔 전력의 감소를 방지하기 위해 여기 파장 0.5 미만에서 샘플 / 기준 화합물의 흡광도를 유지하고, PBD 신호의 선형성을 보장한다. </li></ol> 3. PBD 측정 <ol><li> 참조를 위해 PBD 추적의 측정으로 시작합니다. × 1.0 cm 1.0 cm 또는 1.0 cm × 0.5 cm 석영 셀에 참조 화합물의 솔루션을 배치하고 온도 조절 홀더에 셀을 배치합니다. 두 경로의 길이는 비교 PBD의 진폭을 제공한다. </li><li> 3 ° C.의 온도 증가와 함께 16-35 ° C의 온도 범위에서 온도의 함수로서 상기 기준 PBD 신호를 검출 </li><li> 각 온도 변화에 따라, 위치에 민감한 검출기에 프로브 빔의 위치를​​ 확인하고 다시필요한 경우, 검출기의 중심 위치를 조정한다. </li><li> 식 2에 따른 함수로 [(DN / DT) / C의 P의 ρ] 용어를 PBD 신호의 선형성을 확인합니다. </li><li> 참고 측량 등의 광학 셀의 동일한 방향을 유지하는 기준 화합물과 동일한 광학 셀에 시료 용액을 배치. </li><li> 참고 용으로 동일한 온도 범위 내에서 샘플 PBD 트레이스를 감지하고 [(DN / DT) / C의 P의 ρ] 용어에 대하여 샘플 PBD 진폭의 선형성을 확인. </li></ol> 4. 데이터 분석 편향의 크기 때문에 수학 식 1에 따라 샘플 가열 (ΔV 번째) 및 비열 적 부피 변화 (ΔV의 nonth)에 체적 변화에 정비례한다 : <img src="/files/ftp_upload/50969/50969eq1.jpg" alt="식 (1)" /> 샘플 (S의 진폭) 및 기준 (r)로 PBD 신호는 각각 수학 식 2 및 3을 사용하여 설명 될 수있다. <img src="/files/ftp_upload/50969/50969eq2.jpg" alt="식 2" /> <img src="/files/ftp_upload/50969/50969eq3.jpg" alt="식 3" /> PBD 신호 장비 응답 매개 변수 (K)에 비례하고 아인슈타인의 수는 흡수 된 (E). 식 2의 첫 번째 항은, (DN / DT) (1/ρC의 P) Q 인해 용매에 공개 열에 신호 변화에 해당한다. DN / DT 용어 굴절률의 온도 의존성의 변화를 나타내고, ρ는 용매의 밀도이며, C는 P의 열용량이다. 모든 파라미터 증류수 알려져있다 증류수 및 적절한 버퍼 기준 화합물에 대한 PBD 신호를 비교하여 완충액에 대해 결정될 수있다. Q 열 returne의 양용매 D. ρ (DN / D ρ) 기간은 10 ~ 40 °의 C (10)로부터의 온도 범위에서 온도에 독립적 인 단위가없는 상수이다. Δn ABS 용어 인해 용액의 흡수 종의 존재에 굴절율의 변화에 대응하고, 프로브 광의 파장이 용액 중의 어떤 종의 흡수 스펙트럼에 상대적으로 시프트 된 경우에는 무시할 수이다. 기준 화합물 (R)에서 발생하는 신호는 E H는 ν는 여기 파장에서 광자 에너지 식 3으로 표현된다 E H는 ν = 80.5 만 kcal / 몰 355 nm의 여기를위한. <ol><li> 도 3에서와 같이 사전 트리거 및 포스트 트리거 PBD 신호 간의 차이로서 PBD 기준 신호의 진폭을 가지고 간다. 유사한 방식으로, 고속 (A 발 빠르게)의 진폭을 결정하고 느린 단계 (A 발 느린) 샘플 PBD 신호. </li><li> 악기 응답 매개 변수, K를 제거 기준에 대한 PBD 신호의 진폭에 의해 샘플 PBD 신호의 진폭을 확장하십시오. 기준 신호에 대한 샘플 신호의 비율은 수학 식 4를 부여하고 같이 쓸 수있다 : <img src="/files/ftp_upload/50969/50969eq4.jpg" alt="식 4" /> </li><li> 이 방정식을 사용하여 솔루션 (Q)과 비열 부피 변화 [의 플롯의 각각 기울기와 절편,에서 사진 개시 반응과 관련된 (ΔV의 nonth) (S / R) E에 출시 된 열을 결정 H의 ν] 온도에 따라 기간 [(DN / DT) (1/ρCp)] 대 용어. </li><li> 반응 볼륨과 빠르고 느린 과정에 대한 엔탈피의 변화를 결정하는 식 5-7에 따라 적절한 양자 수율에 관찰 된 볼륨과 엔탈피 변화를 확장하려면. <img sRC = &quot;/ files/ftp_upload/50969/50969eq5.jpg&quot;고도 = &quot;식 5&quot;/&gt; <img src="/files/ftp_upload/50969/50969eq6.jpg" alt="식 (6)" /> <img src="/files/ftp_upload/50969/50969eq7.jpg" alt="식 (7)" /> 동력학은 10 마이크로 초 ~ 200 밀리 초 사이의 시간 스케일로 발생으로 다단계 공정의 경우, 양 및 반응의 각 단계와 관련된 엔탈피 변화가 결정될 수있다. 각 단계에 대한 진폭 및 수명은 볼륨 및 엔탈피 변화의 시간 프로파일을 설명하는 함수 F (t)에 데이터를 피팅에 의해 분석된다. <img alt="식 8" src="/files/ftp_upload/50969/50969eq8.jpg" /> α 0의 빠른에 해당하고 난의 느린 각각의 프로세스에 해당하는 α와 τ는 어디에서 각 반응 단계의 수명입니다. FO 속도 상수의 온도 의존성 가입일R 개별 프로세스가 활성화 엔탈피 및 엔트로피 매개 변수를 쉽게 아 이어링 플롯을 사용하여 측정 할 수있다 (내가 = 1 / τ K). </li></ol>

<ol>
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저자가 공개하는 게 없다.

광열 방법 뒤에 물리적 원리는 광 여기 분자 주위 용매 1,12 열적 가열의 결과, 바닥 상태로 이완 진동을 통해 과량의 에너지를 소산이다. 물과 같은 용매, 이것은 급격한 체적 팽창 (ΔV 번째)를 생성한다. 여기 상태의 분자 인해 결합 벽개 / 형성 및 / 또는 분자 (들) (반 데르 소재 즉 변화의 분자 크기를 변경할 수있는 분자 구조의 변화에 비열 부피 변화 (ΔV의 nonth) 발...

이러한 광 음향 열량, 광열 빔 편향 (PDB) 및 나노초 레이저 여기와 결합 과도 격자로 광열 방법은 일시적인 중간체 1,2의 시간 분해 연구를위한 광학 분광기를 과도 할 수있는 강력한 대안을 나타냅니다. 이러한 과도 흡수 및 IR 분광법 등의 광학 기법과 대조적으로, 그 주위의 발색단의 흡수 변화의 시간 프로파일을 모니터링; 광열 기술은 광학적으로의 시간 프로파일을 조사 할 가치있는 도구를 히트 / 부피 변화의 시간 의존성을 감지하고, 따라서 아르 &quot;침묵&quot;처리합니다. 지금까지, 광 음향 열량 과도 격자가 성공적으로 글로빈 3,4, 산소 센서 단백질과 리간드의 상호 작용에 이원자 리간드의 마이그레이션을 포함하여 사진에 의한 프로세스의 구조적 역학을 연구하기 위해 적용되었습니다 FixL 5, 6 산화 효소 전자와 헴 - 구리의 양자 전송차 광계 II뿐만 아니라 로돕신 7 크립토 8 구조적 역학의 광 이성화. 내부 발색단 및 / 또는 형광 물질이 결여되어 생물학적 시스템에 광열 기술의 애플리케이션을 확장, PBD 기법 따라 광 트리거 갇힌 화합물의 용도 몇 마이크로 초 내에 리간드 / 기질 농도의 증가와 함께 또는 빠른 결합시켰다 갇힌 화합물에. 이 방법은 수있는 리간드 / 기판 내부 형광 또는 발색단 부족한 단백질 및 상업 정지 흐름 악기로 액세스 할 수없는 시간 규모에 바인딩과 관련된 구조적인 변화의 역학과 에너지 부문의 모니터링. 여기 케이지 화합물, 칼슘의 열역학 2 + DM-nitrophen, 사진 - 분열뿐만 아니라 신경 세포의 칼슘 센서의 C-말단 도메인에 칼슘 2 + 협회에 대한 반응 속도 아래로 모니터링 할 PBD의 응용 프로그램스트림 규제 요소 길항제 변조기 (DREAM)가 제공됩니다. CA는 2 +는 10 마이크로 초 이내에 사진 출시 된 CA로부터 2 + DM-nitrophen하고 ~ 300 마이크로 초의 시간 상수 unphotolysed 케이지에 다시 바인딩합니다. 한편, apoDREAM의 존재 밀리 초의 시간 단위로 발생하는 추가적인 운동은 관찰하고 단백질에 결합하는 리간드를 반영한​​다. 생물학적 시스템의 구조적 전환을 조사하는 PBD의 응용 프로그램은 어떻게 든 수단의 어려움으로 인해 제한되었습니다; 강력하고 재현 PBD 신호를 달성하기 위해 프로브와 펌프 빔의 예를 들어 힘든 정렬. 그러나, 계측 셋업의 세심한 디자인, 온도의 정확한 제어, 및 프로브 및 펌프 광의 조심 배향 광범위에 시분 볼륨 및 엔탈피 변화의 모니터링을 허용 일관되고 견고한 PBD 신호를 제공 10 마이크로 초 약 200 밀리 초까지 시간 규모. 또한, modific에서등 동일한 온도, 버퍼 구성, 광 셀 방향, 레이저 파워, 아래 샘플과 참조 흔적의 검출을 보장하기 위해 실험 절차의 관리 포인트는 크게 측정 반응 볼륨과 열량의 실험 오차를 줄일 수 있습니다.

<table border="0" cellpadding="0" cellspacing="0" width="639">
	<colgroup>
		<col />
		<col />
		<col /> 
		<col />
	</colgroup>
	<tbody>
		<tr height="84">
			<td height="84" style="height:84px;width:216px;">1-(4,5-Dimethoxy-2-Nitrophenyl)-1,2-Diaminoethane-N,N,N&#39;,N&#39;-Tetraacetic Acid</td>
			<td style="width:84px;">Life Technologies</td>
			<td style="width:119px;">D-6814</td>
			<td style="width:220px;">DM-nitrophen, cage calcium compound, keep stock solutions in dark to prevent photodissociation,</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:216px;">4-(2-Hydroxyethyl)piperazine-1-ethanesulfonic acid, N-(2-Hydroxyethyl)piperazine-N&#8242;-(2-ethanesulfonic acid)</td>
			<td style="width:84px;">Sigma Adrich</td>
			<td>0909C</td>
			<td>HEPES buffer</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Potassium ferricyanide(III)</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">702587</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;"> </td>
			<td style="width:84px;"> </td>
			<td style="width:119px;"> </td>
			<td style="width:220px;"> </td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Sodium chromate</td>
			<td style="width:84px;">Sigma Aldrich</td>
			<td style="width:119px;">307831</td>
			<td style="width:220px;">reference compound for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">He-Ne Laser Diode 5mW 635nm </td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">54-179</td>
			<td style="width:220px;">use as a probe beam for PBD measurments</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Oscilloscope, </td>
			<td style="width:84px;">LeCroy</td>
			<td style="width:119px;">Wave Surfer 42Xs</td>
			<td style="width:220px;">400 MHz bandwith</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Nd:YAG laser</td>
			<td style="width:84px;">Continuum</td>
			<td style="width:119px;">ML II</td>
			<td>pump beam for PBD measurments</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M355; Nd:YAG laser mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">47-324</td>
			<td>laser mirror for 355 nm laser line</td>
		</tr>
		<tr height="44">
			<td height="44" style="height:44px;width:216px;">M1 and M2; Laser diode mirror</td>
			<td style="width:84px;">Edmund Optics</td>
			<td style="width:119px;">43-532</td>
			<td>visilbe laser flat mirror, wavelength range 300-700 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">P1 and P2; Iris Diaphragm</td>
			<td style="width:84px;">Edmind Optics</td>
			<td style="width:119px;">62-649</td>
			<td style="width:220px;">pin hole to shape the probe and pum beams</td>
		</tr>
		<tr height="84">
			<td height="84" style="height:84px;">L1; bi-convex lens</td>
			<td style="width:84px;">Thorlabs</td>
			<td style="width:119px;">LB1844</td>
			<td style="width:220px;">a lens to focus the probe beam at the detector, EFL 50 mm, wavelength range 350 - 2000 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">DM, dichroic mirror</td>
			<td style="width:84px;">Thorlab </td>
			<td style="width:119px;">DMLP505</td>
			<td style="width:220px;">a longpass dichroic mirror with a cutoff wavelength of 505 nm</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">F1; Edge filter</td>
			<td style="width:84px;">Andower </td>
			<td style="width:119px;">500FH90-25</td>
			<td>a long pass filter with a cutoff wavelength of 500 nm</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:216px;">Temperature-controlled cuvette holder</td>
			<td style="width:84px;">Quantum Northwest</td>
			<td style="width:119px;">FLASH 300</td>
			<td></td>
		</tr>
	</tbody>
</table>

submillisecond conformational changes in proteins resolved by photothermal beam deflection

함께 사진 음향 열량 및 열 격자와 광열 빔 편향은 기존의 정지를 사용하여 액세스 할 수없는 시간 규모를 밀리 초 단위로 마이크로에 단백질의 구조적 변화를 유도 빛의 시간 프로파일 볼륨과 엔탈피 변화를 모니터링 광열 방법의 가족에 속한다 흐름 악기. 양 및 / 또는 전체적인 엔탈피 변화가 프로빙 때문에 또한, 이들 기술은 형광 물질과 또는 발색단 라벨 부족한 단백질 및 다른 생체 고분자에 적용 할 수있다. 칼슘과 관련된 구조적인 변화의 역학과 에너지 부문을 모니터링하려면 + 칼슘 트랜스 듀서, 이러한 신경 세포의 칼슘 센서, 갇힌 칼슘 화합물, DM-nitrophen, 사진 트리거에게 무료로 칼슘의 빠른 (τ &lt;20 마이크로 초) 증가 채용에 바인딩 농도와 연관된 부피 및 엔탈피 변화는 광열 편향 빔 기술을 이용하여 조사됩니다.

PBD의 대표적인 예는 칼슘 2 +의 DM-nitrophen에서 칼슘 2 + 사진 방출에 대한 추적은 그림 3에서 볼 수 있습니다. 느린 단계는 칼슘 2 + nonphotolysed 케이지에 바인딩을 반영하는 반면, 빠른 단계는 칼슘 2 +의 DM-nitrophen 및 CA 2 + 해방의 사진 - 분열에 해당합니다. 온도 의존 인자 [C의 P의 ρ / (DN / DT)]의 함수로서 기준 화합물의 진폭을 확장 빠르고...

Watch this Scientific Journal Video about Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection at JoVE.com

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection | Chemistry | Video

Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

여기에서 우리는 마이크로 및 밀리 역학 및 신경원 칼슘 센서 하류 조절 요소 길항제 변조기 칼슘 연관과 관련된 구조적 변경의 지학을 모니터링 갇힌 칼슘 화합물, DM-nitrophen과 조합 광열 빔 편향 기술의 응용을보고 .

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 ...

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8.9K 조회수.  Florida International University.  여기에서 우리는 마이크로 및 밀리 역학 및 신경원 칼슘 센서 하류 조절 요소 길항제 변조기 칼슘 연관과 관련된 구조적 변경의 지학을 모니터링 갇힌 칼슘 화합물, DM-nitrophen과 조합 광열 빔 편향 기술의 응용을보고 . 

비디오: Submillisecond Conformational Changes in Proteins Resolved by Photothermal Beam Deflection

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 ...

연구

화학

The Use of Gas Chromatography to Analyze Compositional Changes of Fatty Acids in Rat Liver Tissue during Pregnancy

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, yielding results with high accuracy and high reproducibility. In metabolic and nutrition studies GC allows assessment of changes in fatty acid concentrations following interventions or during changes in physiological state such as pregnancy. Solid phase extraction (SPE) using aminopropyl silica cartridges allows separation of the major lipid classes including triacylglycerols, different phospholipids, and cholesteryl esters (CE). GC combined with SPE was used to analyze the changes in fatty acid composition of the CE fraction in the livers of virgin and pregnant rats that had been fed various high and low fat diets. There are significant diet/pregnancy interaction effects upon the omega-3 and omega-6 fatty acid content of liver CE, indicating that pregnant females have a different response to dietary manipulation than is seen among virgin females.

Pregnancy leads to significant changes to the fatty acid composition of maternal tissues. Lipid profiles can be obtained via gas chromatography to allow identification and quantification of fatty acids in individual lipid classes among rats fed various high and low fat diets during pregnancy.

임신 중 쥐의 간 조직에서 지방산의 작곡 변화를 분석하는 가스 크로마토 그래피의 사용

Gas chromatography (GC) is a highly sensitive method used to identify and quantify the fatty acid content of lipids from tissues, cells, and plasma/serum, ...

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 ...

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 ...

Time-resolved Photophysical Characterization of Triplet-harvesting Organic Compounds at an Oxygen-free Environment Using an iCCD Camera

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables the acquisition of photoluminescence spectra covering the time regime from nanoseconds up to 0.1 s. This enables us to follow the changes in the intensity (decay) and emission of the spectra over time. Using this method, it is possible to study diverse photophysical phenomena, such as the emission of phosphorescence, and the contributions of prompt and delayed fluorescence in molecules showing thermally activated delayed fluorescence (TADF). Remarkably, all spectra and decays are obtained in a single experiment. This can be done for solids (thin film, powder, crystal) and liquid samples, where the only limitations are the spectral sensitivity of the camera and the excitation wavelength (532 nm, 355 nm, 337 nm, and 266 nm). This technique is, thus, very important when investigating the excited state dynamics in organic emitters for their application in organic light-emitting diodes and other areas where triplet harvesting is of paramount importance. Since triplet states are strongly quenched by oxygen, emitters with efficient TADF luminescence, or those showing room temperature phosphorescence (RTP), must be correctly prepared in order to remove any dissolved oxygen from solutions and films. Otherwise, no long-lived emission will be observed. The method of degassing solid samples as presented in this work is basic and simple, but the degassing of liquid samples creates additional difficulties and is particularly interesting. A method of minimizing solvent loss and changing the sample concentration, while still enabling to remove oxygen in a very efficient and a repeatable manner, is presented in this work.

Here, we present a method of the spectroscopic characterization of organic molecules by means of time-resolved photoluminescence spectroscopy on the nanosecond-to-millisecond timescale in oxygen-free conditions. Methods to efficiently remove oxygen from the samples and, thus, limit luminescence quenching are also described.

Here, we present a sensible method of the acquisition and analysis of time-resolved photoluminescence using an ultrafast iCCD camera. This system enables ...

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 ...

Co-expression of Multiple Chimeric Fluorescent Fusion Proteins in an Efficient Way in Plants

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent proteins and generation of fluorescent fusion proteins have been wildly used as an effective tool to directly visualize the protein localization and dynamics in cells. It is especially useful to compare them with well-known organelle markers after co-expression with the protein of interest. Nevertheless, classical approaches for protein co-expression in plants usually involve multiple independent expression plasmids, and therefore have drawbacks that include low co-expression efficiency, expression-level variation, and high time expenditure in genetic crossing and screening. In this study, we describe a robust and novel method for co-expression of multiple chimeric fluorescent proteins in plants. It overcomes the limitations of the conventional methods by using a single expression vector that is composed of multiple semi-independent expressing cassettes. Each protein expression cassette contains its own functional protein expression elements, and therefore it can be flexibly adjusted to meet diverse expression demand. Also, it is easy and convenient to perform the assembly and manipulation of DNA fragments in the expression plasmid by using an optimized one-step reaction without additional digestion and ligation steps. Furthermore, it is fully compatible with current fluorescent protein derived bio-imaging technologies and applications, such as FRET and BiFC. As a validation of the method, we employed this new system to co-express fluorescently fused vacuolar sorting receptor and secretory carrier membrane proteins. The results show that their perspective subcellular localizations are the same as in previous studies by both transient expression and genetic transformation in plants.

We have developed a novel method for co-expressing multiple chimeric fluorescent fusion proteins in plants to overcome the difficulties of conventional methods. It takes advantage of using a single expression plasmid that contains multiple functionally independent protein expressing cassettes to achieve protein co-expression.

식물에는 효율적인 방법으로 여러 공상 형광 성 융해 단백질의 공동 식

Information about the spatiotemporal subcellular localization(s) of a protein is critical to understand its physiological functions in cells. Fluorescent ...

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&#8722;

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound electrons with neutral molecules/atoms. State-of-the-art in vacuo anion generation techniques allow application to a broad range of atomic, molecular, and cluster anion systems. These are separated and selected using time-of-flight mass spectrometry. Electrons are removed by linearly polarized photons (photo detachment) using table-top laser sources which provide ready access to excitation energies from the infra-red to the near ultraviolet. Detecting the photoelectrons with a velocity mapped imaging lens and position sensitive detector means that, in principle, every photoelectron reaches the detector and the detection efficiency is uniform for all kinetic energies. Photoelectron spectra extracted from the images via mathematical reconstruction using an inverse Abel transformation reveal details of the anion internal energy state distribution and the resultant neutral energy states. At low electron kinetic energy, typical resolution is sufficient to reveal energy level differences on the order of a few millielectron-volts, i.e., different vibrational levels for molecular species or spin-orbit splitting in atoms. Photoelectron angular distributions extracted from the inverse Abel transformation represent the signatures of the bound electron orbital, allowing more detailed probing of electronic structure. The spectra and angular distributions also encode details of the interactions between the outgoing electron and the residual neutral species subsequent to excitation. The technique is illustrated by the application to an atomic anion (F&#8722;), but it can also be applied to the measurement of molecular anion spectroscopy, the study of low lying anion resonances (as an alternative to scattering experiments) and femtosecond (fs) time resolved studies of the dynamic evolution of anions.

Photoelectron Imaging of Anions Illustrated by 310 Nm Detachment of F&lt;sup&gt;&amp;#8722;&lt;/sup&gt;

Here, we present a protocol for photoelectron imaging of anionic species. Anions generated in vacuo and separated by mass spectrometry are probed using velocity mapped photoelectron imaging, providing details of anion and neutral energy levels, anion and neutral structure and the nature of the anion electronic state.

F− 의 310 Nm 분리에 의해 설명 하는 음이온의 광전자 이미징

Anion photoelectron imaging is a very efficient method for the study of energy states of bound negative ions, neutral species and interactions of unbound ...

Improving High Viscosity Extrusion of Microcrystals for Time-resolved Serial Femtosecond Crystallography at X-ray Lasers

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray free electron lasers (XFELs). A series of experiments using the light-driven proton pump bacteriorhodopsin have further established these injectors as a preferred option to deliver crystals for time-resolved serial femtosecond crystallography (TR-SFX) to resolve structural changes of proteins after photoactivation. To obtain multiple structural snapshots of high quality, it is essential to collect large amounts of data and ensure clearance of crystals between every pump laser pulse. Here, we describe in detail how we optimized the extrusion of bacteriorhodopsin microcrystals for our recent TR-SFX experiments at the Linac Coherent Light Source (LCLS). The goal of the method is to optimize extrusion for a stable and continuous flow while maintaining a high density of crystals to increase the rate at which data can be collected in a TR-SFX experiment. We achieve this goal by preparing lipidic cubic phase with a homogenous distribution of crystals using a novel three-way syringe coupling device followed by adjusting the sample composition based on measurements of the extrusion stability taken with a high-speed camera setup. The methodology can be adapted to optimize the flow of other microcrystals. The setup will be available for users of the new Swiss Free Electron Laser facility.

The success of a time-resolved serial femtosecond crystallography experiment is dependent on efficient sample delivery. Here, we describe protocols to optimize the extrusion of bacteriorhodopsin microcrystals from a high viscosity micro-extrusion injector. The methodology relies on sample homogenization with a novel three-way coupler and visualization with a high-speed camera.

X 선 레이저에서 직렬 펨 결정학 시간 해결에 대 한 Microcrystals의 고 점도 압출을 개선

High-viscosity micro-extrusion injectors have dramatically reduced sample consumption in serial femtosecond crystallographic experiments (SFX) at X-ray ...