우리는 올리버 Brüstle 및 줄기 세포 공학 그룹, 지원 및 귀중한 토론 본 대학의 모든 구성원을 감사드립니다. 우리는 SDS - PAGE 및 프로젝트 전반에 걸쳐 지속적인 지원의 준비에 대한 사빈 쉥크 감사합니다. 니콜 러스와 안나 Magerhans 뛰어난 기술 지원을 제공했습니다. 또한, 우리는 영화의 생산을위한 안드레아스 바, 쉴라 머텐스 감사하고 싶습니다. 이 작품은 폭스바겐 재단 (AZ I/77864) 및 교육과 연구의 독일 교육부 (BMBF, 01 GN 0813)에서 보조금에 의해 지원되었다.

발현 벡터와 표현의 건설 : pSESAME - Cre 호텔 표현 벡터는 표준 복제 방법을 사용 AvrII 및 NheI 제한 사이트를 통해 pSESAME에 Cre 호텔 - 인코딩 조각을 삽입하여 건설되었습니다. pSESAME은 히스티딘 태그, 두드리는 소리 - 도메인, NLS 시퀀스와 Cre 호텔, 약식 HTNCre 구성된 융합 단백질을 인코딩합니다. HTNCre 표현에 대한 pSESAME - Cre 호텔은 튜너 (DE3) pLacI로 변환 및 글리세롤 주식을 준비하는 데 사용되었다. <ol><li> 이상 박 문화 글리세롤 주식의 변형 박테리아와 코팅 pipet 팁을 사용하여 주사를했다. 이상의 밤 문화는 0.5 %의 포도당과 보충 LB 미디어로 구성되어 [V / V] 50 μg / ML의 최종 농도에 carbenicillin 37에서 성장을 허용했다 ° 16시간를위한 C. </li><li> 다음날 밀도 성장을 통해 밤 문화은 1 40 비율로 표현 문화를 예방하는 데 사용되었으며 37 ° C.에서 인큐베이터에 넣어되었습니다 표현 문화는 100 μg / ML의 최종 농도에서 0.5 % 포도당 [V / V]와 암피실린로 보충 TB 미디어 구성되어. </li><li> 1.5 OD 595에서 표현 문화는 1 H.위한 0.5 MM의 IPTG로 유도했습니다 </li><li> 후 박테리아는 SLA3000 로터에서 10 분 동안 5,000 rpm으로 원심 분리에 의해 수집되었다. </li><li> 박테리아의 알약은 마이너스 20 ° C 정화까지 저장되었습니다. </li></ol> 세포 투과 단백질의 정제 : <ol><li> 냉동 세균 알약은 실온에서 15 분 동안 리터 플라스크 문화 당 10 ML의 용해 버퍼에서 resuspended되었습니다. </li><li> 실온에서 혼합하는 동안 정지 후 추가로 15 분 동안 1 MG / ML 라이 소 자임와 incubated했다. </li><li> 25 U / ML benzonase는 나중에 추가하고 실온에서 15 분 동안 혼합하는 동안 incubated되었습니다. </li><li> 전력의 45 %로 0.5 S 펄스와 1.5 분 얼음 sonification 후, 1 ML 차가운 tartaric 소금 버퍼 (TSB)가 당 ML 정지 신중하면서 추가되었습니다 혼합하고 얼음에서 5 분 incubated. lysate 분수 (L)의 SDS - PAGE 샘플 찍은. </li><li> 걷어 lysate는 ° C 30 분 30,000 G. 4 원심 분리하여 얻은 가용성 (S)과 불용성 분수 (I)의 SDS - PAGE 샘플가 함락되었다. </li><li> 뜨는은 신선한 50 ML 팔콘 튜브 안으로 옮겨졌 다가 그리고 부드럽게 4 1 H에 대한 혼합되었습니다 ° C 50 % 초기 표현 문화의 리터 당 니켈 - NTA 슬러리 2 ML과 함께. </li><li> 현탁액은 중력 흐름 EconoPac 칼럼 (분율 흐름을 통해 (FT) 찍은의 SDS - PAGE 샘플)로 포장 및 세척 버퍼 5 침대 볼륨과 함께 두 번 씻어했다. 모두 세척 분수 (W1 &amp; W2)의 SDS - PAGE 샘플을 채취했다. </li><li> HTNCre 함유 분수는 SDS - PAGE 분석을위한 eluate 분율 (E)의 용출 버퍼와 샘플 3 침대 볼륨 eluted되었습니다 것은 찍은. </li><li> 이미다졸 두 번이나 높은 소금 버퍼에 대한 용출 분획을 dialyzing에 의해 제거되었습니다. </li><li> 단백질 솔루션은 더욱 두 번 글리세롤 버퍼에 대한 dialyzing로 집중되었다. 모든 투석 단계에서 샘플로 버퍼의 비율은 최소한 50되었다. 이 절차는 200 450 μm의 사이에 평소와 농도에 HTNCre를 포함하는 글리세롤 주식 솔루션으로 이어지는, 즉 표현 문화 1 리터 단백질 ~ 12 MG가 발생합니다. SDS - PAGE 분석을위한 글리세롤 주식 (GS)의 샘플을 수집했다. HTNCre 주식 솔루션은 마이너스 20 저장할 수 있습니다 ° C. </li></ol><img border="0" src="/files/ftp_upload/1627/1627fig1tmb.jpg" alt="그림 1" /> 그림 1 : Cre 호텔 recombinase의 정화 과정을 통해 수집된 샘플의 SDS - PAGE 분석. Cre 호텔 표현의 유도는 lysate 소수의 지배적인 밴드로 표시됩니다. 단백질의 일부​​는 불용성이지만 eluate과 글리세롤 주식 분수 모습으로 Cre 호텔 단백질이 더 풍부하게하실 수 있습니다. L : Lysate, I : 불용성, S : 뜨는, FT : 흐름 -을 통해, W : 세탁, E : Eluate, GS : Gylcerol 주식. 하시기 바랍니다 <a href="https://www.jove.com/files/ftp_upload/1627/1627fig1.jpg">여기를 클릭</a> 그림 1의 더 큰 버전을 볼 수 있습니다. murine 배아 줄기 (ES) 세포에 단백질 전달 : <ol><li> 조건부 β - Galactosidase 기자 다섯 구조를 갖고 ES 세포가 자기편 세포의 분리에 대한 TrypLE ™ Express를 사용하여 단일 세포로 씨앗을 품고 있었다. 4~6시간 후 세포가 다시 연결했고 매체가 삭제되었습니다. </li><li> 이후 ES 세포는 16 시간 동안 HTNCre 함유 매체와 incubated했다. <ul><li> 글리세롤의 품절 HTNCre 단백질의 적절한 금액 (10 μm의에 해당)은 ES 매체와 이후 살균 filtrated (0.22 μm의)에 희석되었다. </li></ul></li><li> 단백질 전달 매체는 정상적인 성장 매체로 변경되었습니다 후. </li><li> 이틀 세포는 PBS로 씻어 10 분 4 % Paraformaldehyde (PFA)가 수정되었습니다 후에. </li><li> 두 addiX - 걸의 얼룩이 수행되기 전에 PBS로 세척 tional 단계를 수행했다. </li><li> 고정 세포 X - 걸의 얼룩 솔루션 6 층 덮여 37 ° C. 밤 동안 incubated되었습니다 </li></ol> 대표 결과 : 다음 날 X - 걸의 얼룩 솔루션은 했잖아요되었으며 세포는 현미경 분석을위한 PBS의 레이어로 쓰여져있었습니다. recombined 세포의 80-100%는 β - Galactosidase의 활동에 의해 판단 murine ES 세포 내에서 관찰 수 있습니다.

<ol>
	<li>Gump, J. M., Dowdy, S. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=TAT+transduction:+the+molecular+mechanism+and+therapeutic+prospects.">TAT transduction: the molecular mechanism and therapeutic prospects.</a> Trends Mol Med. 13 (10), 443-448 (2007).</li><li>Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+transduction+revisited:+novel+insights+into+the+mechanism+underlying+intracellular+delivery+of+proteins.">Protein transduction revisited: novel insights into the mechanism underlying intracellular delivery of proteins.</a> Curr Pharm Des. 14 (34), 3628-3636 (2008).</li><li>Branda, C. S., Dymecki, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Talking+about+a+revolution:+The+impact+of+site-specific+recombinases+on+genetic+analyses+in+mice.">Talking about a revolution: The impact of site-specific recombinases on genetic analyses in mice.</a> Dev Cell. 6 (1), 7-28 (2004).</li><li>Nolden, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Site-specific+recombination+in+human+embryonic+stem+cells+induced+by+cell-permeant+Cre+recombinase.">Site-specific recombination in human embryonic stem cells induced by cell-permeant Cre recombinase.</a> Nat Methods. 3 (6), 461-467 (2006).</li><li>Zhang, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Inducible+site-directed+recombination+in+mouse+embryonic+stem+cells.">Inducible site-directed recombination in mouse embryonic stem cells.</a> Nucleic Acids Res. 24 (4), 543-548 (1996).</li><li>Peitz, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+purification+of+cell-permeant+Cre+and+germline+transmission+after+transduction+into+mouse+embryonic+stem+cells.">Enhanced purification of cell-permeant Cre and germline transmission after transduction into mouse embryonic stem cells.</a> Genesis. 45 (8), 508-517 (2007).</li><li>Bosnali, M., Edenhofer, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Generation+of+transducible+versions+of+transcription+factors+Oct4+and+Sox2.">Generation of transducible versions of transcription factors Oct4 and Sox2.</a> Biol Chem. 389 (7), 851-861 (2008).</li><li>Landry, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Runx+genes+are+direct+targets+of+Scl/Tal1+in+the+yolk+sac+and+fetal+liver.">Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver.</a> Blood. 111 (6), 3005-3014 (2008).</li></ol>

Cre 호텔 융합 단백질의 정화 과정에서 그것은 원심 분리하기 전에 얼음 차가운 TBS 버퍼의 추가를 생략하지 않는 것이 중요합니다. 그렇지 않으면 Cre 호텔 recombinase는 글리세롤 버퍼 내에 침전하는 경향이있다. eluate 분율이 될 나타나는 경우 솔루션을 다시 해제 때까지 융합 단백질 추가 용출 버퍼의 높은 농도로 인해 흐린이 추가되어야합니다. Cre 호텔 융합 단백질의 10 μm의의 응용 프로그램은 일반적으로 80-100%의 재조합 효율을 초래합니다. 소태아 세럼 (FCS)는 ES 세포 매체의 주요 구성 요소가되는 것은 강력하게 단백질 전달을 억제. Cre 호텔 recombinase의 따라서 고농도 사용할 수 있었다. 혈청이없는 조건에서 작업할 때 더 적은 단백질 (0.5-2 μm의)는 유사한 재조합 효율을 달성하는 데 사용할 수 있습니다. 손에 pSESAME 벡터 시스템과 하나가 같은 Oct4와 Sox2 7 Scl/Tal1 8로 전사 요인 등 다른 단백질로 단백질 전달의 기법을 적용할 수 있습니다.

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		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>TUNER (DE3) pLacI</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>70625</td>
<td>&nbsp;</td>
<tr>
<td>Glycerol</td>
<td>&nbsp;</td>
<td>Carl Roth</td>
<td>3783.2</td>
<td>&nbsp;</td>
<tr>
<td>Na2HPO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>T876.1</td>
<td>&nbsp;</td>
<tr>
<td>Trizma Base</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>T1503</td>
<td>&nbsp;</td>
<tr>
<td>HCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>4625.1</td>
<td>&nbsp;</td>
<tr>
<td>Imidazol</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>X998.4</td>
<td>&nbsp;</td>
<tr>
<td>NaCl</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>9265.2</td>
<td>&nbsp;</td>
<tr>
<td>Yeast Extract</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>2363.4</td>
<td>&nbsp;</td>
<tr>
<td>Trypton/Pepton</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>8952.4</td>
<td>&nbsp;</td>
<tr>
<td>K2HPO4 </td>
<td>&nbsp;</td>
<td>Roth</td>
<td>P749.2</td>
<td>&nbsp;</td>
<tr>
<td>KH2PO4</td>
<td>&nbsp;</td>
<td>Roth</td>
<td>3904.1</td>
<td>&nbsp;</td>
<tr>
<td>Ampicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A9518</td>
<td>&nbsp;</td>
<tr>
<td>Carbenicillin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>6344.2</td>
<td>&nbsp;</td>
<tr>
<td>HEPES</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>H3375</td>
<td>&nbsp;</td>
<tr>
<td>Lysozyme</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>62971</td>
<td>&nbsp;</td>
<tr>
<td>Benzonase</td>
<td>&nbsp;</td>
<td>Novagen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>L-Tartaric acid, disodium salt </td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>50% Ni-NTA slurry</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>R901-15</td>
<td>&nbsp;</td>
<tr>
<td>EconoPac columns</td>
<td>&nbsp;</td>
<td>Biorad</td>
<td>732-1010</td>
<td>&nbsp;</td>
<tr>
<td>Sterile filter 0,22&mu;m</td>
<td>&nbsp;</td>
<td>Whatman</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Paraformaldehyde (PFA)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>LB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, NaCl</td>
</tr>
<tr>
<td>TB medium</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>Yeast extract, Trypton/Pepton, Glycerol, K2HPO4, KH2PO4</td>
</tr>
<tr>
<td>Lysis Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50 mM Na2HPO4, 5 mM Tris, pH 7.8</td>
</tr>
<tr>
<td>Tartaric Salt Buffer (TSB)</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB containing 2 M L-Tartaric acid, disodium salt, and 20 mM Imidazol</td>
</tr>
<tr>
<td>Washing Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 15 mM Imidazol</td>
</tr>
<tr>
<td>Elution Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>PTB, 500 mM NaCl, 250 mM Imidazol</td>
</tr>
<tr>
<td>High Salt Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>600 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>Gylcerol Buffer</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>50% glycerol, 500 mM NaCl, 20 mM HEPES, pH 7.4</td>
</tr>
<tr>
<td>TrypLE&trade; Express</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>ESGRO (LIF)</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>NEAA</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11140035</td>
<td>&nbsp;</td>
<tr>
<td>L-Glutamin</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>25030024</td>
<td>&nbsp;</td>
<tr>
<td>&beta;-Mercaptoethanol</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>31350010</td>
<td>&nbsp;</td>
<tr>
<td>DMEM</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>11960044</td>
<td>&nbsp;</td>
<tr>
<td>PBS</td>
<td>&nbsp;</td>
<td>Gibco</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>Fetal Calf Serum (FCS)</td>
<td>&nbsp;</td>
<td>PAA</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal staining solution:</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>4 mM K3(FeIII(CN)6), 4 mM K4(FeII(CN)6), 2mM MgCl2 0.4 mg/mL X-Gal solved in PBS</td>
</tr>
<tr>
<td>K3(FeIII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-3367</td>
<td>&nbsp;</td>
<tr>
<td>K4 (FeII(CN)6)</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>P-9387</td>
<td>&nbsp;</td>
<tr>
<td>MgCl2</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>&nbsp;</td>
<tr>
<td>X-Gal</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>B4252</td>
<td>&nbsp;</td>		</tbody>
		</table>
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engineering cell-permeable protein

단백질 전달 기술은 포유 동물 세포에 생물 학적 활성 물질의 직접 전송을 가능하게 [검토 1,2 참조]를. 이것은 소위 세포 관통 펩티드 (CPPs)의 translocating 능력을 사용 할 수 들면, 또한 단백질 전달 도메인 (PTDs)로 지정. 인간 면역 결핍 바이러스 1 형 (HIV - 1) 태트 (전사의 횡단 활성제) 단백질이 널리 사용되고 있습니다 파생된 문신 - CPP. 적극적으로 청구 문신함으로써 endocytosis 또는 / 및 직접 멤브레인 보급률 2 세포 멤브레인의 장벽을 극복 세포 투과성을 촉진합니다. 핵 지방화 신호와 함께 (NLS) 융합 단백질은 핵 전시 기능을 입력할 수 있습니다. 우리의 비디오 프레 젠 테이션 세포 투과 단백질 공학, 건설, 생산 및 DNA - 수정 효소 Cre 호텔의 세포 투과 버전의 응용 프로그램에 대한 예증으로 보여줍니다. CRE는 체외에서 생체내의 포유 동물 세포의 34 기본 쌍의 loxP 사이트를 인식하고 재결합 수있는 특정 사이트 recombinase입니다. 따라서 Cre 호텔 / loxP 시스템은 널리 조건 살아있는 세포 3,4의 게놈의 변이를 유도하는 데 사용됩니다. 세포 활성 Cre 호텔 recombinase의 배달 그러나, 제한을 나타냅니다. 우리는 유전자 수준의 관심을 직접 삽입이 가능하고 신속하게 편리하고 표준화된 방식으로 벡터 내에서 사용되는 다른 도메인과 태그를 복제하는 플랫폼을 제공 pSESAME 벡터 시스템을 설명합니다. 다른 태그 재정렬하면 높은 수율과 더 나은 용해도를 달성하는 가능성을 제공하는 융합 단백질의 생화 학적 속성을 수정 표시되었습니다. 우리와 E. 대장균에서 재조합 세포 permeant 단백질을 표현하고 정화하는 방법을 보여줍니다. 재조합 단백질 Cre 호텔의 기능은 마침내 세포 recombinase 활동을 평가하여 세포 배양에 확인됩니다.

Watch this Scientific Journal Video about Engineering Cell-permeable Protein at JoVE.com

Engineering Cell-permeable Protein

단백질 전달은 세포에 생물학적으로 활성화된 단백질의 직접 배달을 수 있습니다. 이러한 DNA의 transfection 또는 바이러스성 도입으로 종래의 방법과는 달리이 비침습 패러다임은 세포 독성 및 영구 유전자 조작에 의해 종양 발생 변화의 위험을 우회 titratable 방식으로 고효율 세포 조작이 가능합니다.

공학 세포 투과 단백질

The protein transduction technique enables the direct delivery of biologically active material into mammalian cells [for review see 1,2]. For this one can ...

Research

JoVE Journal

Biology

14.4K 조회수.  University of Bonn - Life & Brain Center and Hertie Foundation. 단백질 전달은 세포에 생물학적으로 활성화된 단백질의 직접 배달을 수 있습니다. 이러한 DNA의 transfection 또는 바이러스성 도입으로 종래의 방법과는 달리이 비침습 패러다임은 세포 독성 및 영구 유전자 조작에 의해 종양 발생 변화의 위험을 우회 titratable 방식으로 고효율 세포 조작이 가능합니다.

비디오: Engineering Cell-permeable Protein

Experimental Approaches to Tissue Engineering

조직 공학 실험 접근법

연구

생물학

Micro-scale Engineering for Cell Biology

세포 생물학을위한 미세 규모 공학

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

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

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

유기 용매 및 초산없이 쿠매시 G - 250로 젤의 단백질 염색법

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

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering 

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development of constructs for tissue engineering. This environment better mimics what cells observe in vivo, compared to standard tissue culture, due to the tissue-like properties and 3D environment. Synthetic polymeric hydrogels are water-swollen networks that can be designed to be stable or to degrade through hydrolysis or proteolysis as new tissue is deposited by encapsulated cells. A wide variety of polymers have been explored for these applications, such as poly(ethylene glycol) and hyaluronic acid. Most commonly, the polymer is functionalized with reactive groups such as methacrylates or acrylates capable of undergoing crosslinking through various mechanisms. In the past decade, much progress has been made in engineering these microenvironments - e.g., via the physical or pendant covalent incorporation of biochemical cues - to improve viability and direct cellular phenotype, including the differentiation of encapsulated stem cells (Burdick et al.).
The following methods for the 3D encapsulation of cells have been optimized in our and other laboratories to maximize cytocompatibility and minimize the number of hydrogel processing steps. In the following protocols (see Figure 1 for an illustration of the procedure), it is assumed that functionalized polymers capable of undergoing crosslinking are already in hand; excellent reviews of polymer chemistry as applied to the field of tissue engineering may be found elsewhere (Burdick et al.) and these methods are compatible with a range of polymer types. Further, the Michael-type addition (see Lutolf et al.) and light-initiated free radical (see Elisseeff et al.) mechanisms focused on here constitute only a small portion of the reported crosslinking techniques. Mixed mode crosslinking, in which a portion of reactive groups is first consumed by addition crosslinking and followed by a radical mechanism, is another commonly used and powerful paradigm for directing the phenotype of encapsulated cells (Khetan et al., Salinas et al.).

Cellular Encapsulation in 3D Hydrogels for Tissue Engineering

We present protocols for the 3-dimensional (3D) encapsulation of cells within synthetic hydrogels. The encapsulation procedure is outlined for two commonly used methods of crosslinking (michael-type addition and light-initiated free radical mechanisms), as well as a number of techniques for assessing encapsulated cell behavior.

조직 공학을위한 3D Hydrogels에서 셀룰러 봉지

The 3D encapsulation of cells within hydrogels represents an increasingly important and popular technique for culturing cells and towards the development ...

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

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

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

액상 복구와 분자량 기반 분류에 대한 GELFREE 8100 분획화 시스템을 사용하여

The GELFREE 8100 Fractionation System is a novel protein fractionation system designed to maximize protein recovery during molecular weight based ...

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific properties, such as binding affinity, agonist or antagonist behavior, or stability, relative to the native sequence. Protein design lies at the center of current advances drug design and discovery. Not only does protein design provide predictions for potentially useful drug targets, but it also enhances our understanding of the protein folding process and protein-protein interactions. Experimental methods such as directed evolution have shown success in protein design. However, such methods are restricted by the limited sequence space that can be searched tractably. In contrast, computational design strategies allow for the screening of a much larger set of sequences covering a wide variety of properties and functionality. We have developed a range of computational de novo protein design methods capable of tackling several important areas of protein design. These include the design of monomeric proteins for increased stability and complexes for increased binding affinity.
To disseminate these methods for broader use we present Protein WISDOM (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>), a tool that provides automated methods for a variety of protein design problems. Structural templates are submitted to initialize the design process. The first stage of design is an optimization sequence selection stage that aims at improving stability through minimization of potential energy in the sequence space. Selected sequences are then run through a fold specificity stage and a binding affinity stage. A rank-ordered list of the sequences for each step of the process, along with relevant designed structures, provides the user with a comprehensive quantitative assessment of the design. Here we provide the details of each design method, as well as several notable experimental successes attained through the use of the methods.

Protein WISDOM: A Workbench for In silico De novo Design of BioMolecules

We developed computational de novo protein design methods capable of tackling several important areas of protein design. To disseminate these methods we present Protein WISDOM, an online tool for protein design (<a href="http://www.proteinwisdom.org" target="_blank">http://www.proteinwisdom.org</a>). Starting from a structural template, design of monomeric proteins for increased stability and complexes for increased binding affinity can be performed.

단백질 지혜 : 용 워크 벤치<em&gt; 실리코에</em&gt;<em&gt; 드 노보</em생체 분자의&gt; 디자인

The aim of de novo protein design is to find the amino acid sequences that will fold into a desired 3-dimensional structure with improvements in specific ...

Mouse Genome Engineering Using Designer Nucleases

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well as for modeling human diseases and testing therapeutic strategies. Recent advances in the use of designer nucleases such as zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) 9 system for site-specific genome engineering open the possibility to perform rapid targeted genome modification in virtually any laboratory species without the need to rely on embryonic stem (ES) cell technology. A genome editing experiment typically starts with identification of designer nuclease target sites within a gene of interest followed by construction of custom DNA-binding domains to direct nuclease activity to the investigator-defined genomic locus. Designer nuclease plasmids are in vitro transcribed to generate mRNA for microinjection of fertilized mouse oocytes. Here, we provide a protocol for achieving targeted genome modification by direct injection of TALEN mRNA into fertilized mouse oocytes.

Designer nucleases such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) can be used to modify the genome of mouse preimplantation embryos by triggering both the nonhomologous end joining (NHEJ) and homologous recombination (HR) pathways. These advances enable the rapid generation of mice with precise genetic modifications.

디자이너 클레아 제를 사용하여 마우스 게놈 엔지니어링

Transgenic mice carrying site-specific genome modifications (knockout, knock-in) are of vital importance for dissecting complex biological systems as well ...

siRNA Screening to Identify Ubiquitin and Ubiquitin-like System Regulators of Biological Pathways in Cultured Mammalian Cells

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that regulates diverse biological pathways including the hypoxia response, proteostasis, the DNA damage response and transcription.&#160; To better understand how UBLs regulate pathways relevant to human disease, we have compiled a human siRNA &#8220;ubiquitome&#8221; library consisting of 1,186 siRNA duplex pools targeting all known and predicted components of UBL system pathways. This library can be screened against a range of cell lines expressing reporters of diverse biological pathways to determine which UBL components act as positive or negative regulators of the pathway in question.&#160; Here, we describe a protocol utilizing this library to identify ubiquitome-regulators of the HIF1A-mediated cellular response to hypoxia using a transcription-based luciferase reporter.&#160; An initial assay development stage is performed to establish suitable screening parameters of the cell line before performing the screen in three stages: primary, secondary and tertiary/deconvolution screening. &#160;The use of targeted over whole genome siRNA libraries is becoming increasingly popular as it offers the advantage of reporting only on members of the pathway with which the investigators are most interested.&#160; Despite inherent limitations of siRNA screening, in particular false-positives caused by siRNA off-target effects, the identification of genuine novel regulators of the pathways in question outweigh these shortcomings, which can be overcome by performing a series of carefully undertaken control experiments.

Here, we describe a methodology to perform a targeted siRNA &#8220;ubiquitome&#8221; screen to identify novel ubiquitin and ubiquitin-like regulators of the HIF1A-mediated cellular response to hypoxia.&#160; This can be adapted to any biological pathway where a robust read out of reporter activity is available.

배양 된 포유 동물 세포의 생물 학적 경로의 유비퀴틴과 유비​​퀴틴 같은 시스템 레귤레이터를 확인하는 siRNA를 상영

Post-translational modification of proteins with ubiquitin and ubiquitin-like molecules (UBLs) is emerging as a dynamic cellular signaling network that ...

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and the inherent instability of many membrane proteins once solubilized in detergents. A protocol is described that combines ligation independent cloning of membrane proteins as GFP fusions with expression in Escherichia coli detected by GFP fluorescence. This enables the construction and expression screening of multiple membrane protein/variants to identify candidates suitable for further investment of time and effort. The GFP reporter is used in a primary screen of expression by visualizing GFP fluorescence following SDS polyacrylamide gel electrophoresis (SDS-PAGE). Membrane proteins that show both a high expression level with minimum degradation as indicated by the absence of free GFP, are selected for a secondary screen. These constructs are scaled and a total membrane fraction prepared and solubilized in four different detergents. Following ultracentrifugation to remove detergent-insoluble material, lysates are analyzed by fluorescence detection size exclusion chromatography (FSEC). Monitoring the size exclusion profile by GFP fluorescence provides information about the mono-dispersity and integrity of the membrane proteins in different detergents. Protein: detergent combinations that elute with a symmetrical peak with little or no free GFP and minimum aggregation are candidates for subsequent purification. Using the above methodology, the heterologous expression in E. coli of SED (shape, elongation, division, and sporulation) proteins from 47 different species of bacteria was analyzed. These proteins typically have ten transmembrane domains and are essential for cell division. The results show that the production of the SEDs orthologues in E. coli was highly variable with respect to the expression levels and integrity of the GFP fusion proteins. The experiment identified a subset for further investigation.

Green Fluorescent Protein-based Expression Screening of Membrane Proteins in Escherichia coli

A streamlined approach to screening for the expression of recombinant membrane proteins in Escherichia coli based on fusion to green fluorescent protein is presented.

막 단백질의 녹색 형광 단백질 기반의 표현 상영에<em&gt; 대장균</em

The production of recombinant membrane proteins for structural and functional studies remains technically challenging due to low levels of expression and ...

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged as useful tools for targeted genome engineering. Like many other DNA-binding proteins, zinc-fingers also possess the innate ability to cross cell membranes. We recently demonstrated that this intrinsic cell-permeability could be leveraged for intracellular protein delivery. Genetic fusion of zinc-finger motifs leads to efficient transport of protein and enzyme cargo into a broad range of mammalian cell types. Unlike other protein transduction technologies, delivery via zinc-finger domains does not inhibit enzyme activity and leads to high levels of cytosolic delivery. Here a detailed step-by-step protocol is presented for the implementation of zinc-finger technology for protein delivery into mammalian cells. Key steps for achieving high levels of intracellular zinc-finger-mediated delivery are highlighted and strategies for maximizing the performance of this system are discussed.

Direct Protein Delivery to Mammalian Cells Using Cell-permeable Cys2-His2 Zinc-finger Domains

Zinc-finger domains are intrinsically cell-permeable and capable of mediating protein delivery into a broad range of mammalian cell types. Here, a detailed step-by-step protocol for implementing zinc-finger technology for intracellular protein delivery is presented.

세포 투과성의 Cys를 사용하여 포유 동물 세포에 직접 단백질 배달<sub&gt; 2</sub&gt; - 그의<sub&gt; 2</sub&gt; 아연 손가락 도메인

Due to their modularity and ability to be reprogrammed to recognize a wide range of DNA sequences, Cys2-His2 zinc-finger DNA-binding domains have emerged ...