1. 샘플 준비 서양은 모래 바닥의 첫 번째 단계는 샘플 준비합니다. 젤 세포와 조직을 실행하기위한 샘플을 준비하기 위해서는 관심의 단백질을 공개 lysed해야합니다. <ul><li> 다목적 및 가용성 단백질 추출에 사용하기 쉬운 편리 즉시 사용 시약은 CytoBuster 또는 PhosphoSafe입니다. 이러한 추출 버퍼는 포유 동물 및 곤충 세포에서 가용성 단백질의 효율적인 추출에 최적화된 세제를 포함하고 있습니다. CytoBuster 단백질 추출 시약의 부드러운 아닌 이온 성분은 sonication이나 동결 / 해동 보조 치료를 위해 필요없이 기능 활성화 내생이나 표현 단백질의 분리 수 있습니다. PhoshoSafe 네 인산 가수 분해 효소 억제제를 포함하는의 추가 혜택을했다. <ol><li> 저속 원심 분리 (2,500 XG의 예 5 분)에 의해 펠렛 세포가 잘 세포 펠렛을 물기. </li><li> 10 6 셀 당 150 μL (셀 크기에 따라 달라질 수 있습니다 CytoBuster 단백질 추출 시약의 최적의 금액)을 사용하여 CytoBuster 단백질 추출 시약으로 세포를 Resuspend. </li><li> 5 분 실온에서 알을 품다. </li><li> 적합한 튜브로 전송하고 4 16,000 XG에서 5 분 스핀 ° C. </li><li> 전송 새로운 튜브로 (세포 추출물) 뜨는 취소 및 분석 진행합니다. </li></ol></li><li> 전문 단백질 추출을 위해, 우리는 고품질 ProteoExtract 키트를 사용하는 것이 좋습니다. EMD는 mitochondrial 고립, subcellular 프로테옴 추출, transmembrane 단백질의 추출, 그리고 많은 다른 이상 한 다스 키트가 있습니다. 자세한 내용은 우리가 서양 얼룩의 방문 페이지를 방문하십시오. </li><li> EMD는 또한 프로 테아제와 인산 가수 분해 효소 억제제 칵테일 세트의 다양한 판매하고 있습니다. 즉시 용해가 발생로서, proteolysis, dephosphorylation 및 변성이 시작됩니다. 샘플 얼음이나 4에 보관하는 경우 이러한 이벤트는 엄청난 속도가 느려졌 수 있습니다 ° 항상 적절한 억제제에 C가 용해 버퍼에 새로운 추가됩니다. 자세한 내용은 우리가 서양 얼룩의 방문 페이지를 방문하십시오. </li></ul> 2. SDS - PAGE 모래 바닥 서양 두 번째 단계는 단백질 분리됩니다. 단백질은 분자량에 따라 구분됩니다. <ul><li> 당신은 자신의 페이지 젤을 할 수 있지만 우리는 상업적으로 사전 캐스트 젤을 사용하는 것입니다. <ol><li> 샘플 준비 후 단백질 농도를 결정하기 위해 준비가되어 있습니다. EMD는 농도를 측정하는이 두 키트를 가지고, 그 중 하나가 아닌 방해하는 단백질 분석 키트 및 BCA 단백질 분석 키트되고있는 다른되지. </li><li> 단백질 농도가 평가되면 우리는 젤 로딩을위한 샘플을 준비하는 준비가되어 있습니다. 항체는 일반적으로 관심의 단백질의 작은 부분을 (에피토프라고도 함)임을 인식하고이 도메인은 단백질의 3 차원 형태 내에 있습니다. 그것은 단백질을 펼쳐 필요가이 부분에 대한 항체의 액세스를 설정하려면 다음과 같이하십시오. </li><li> 변성하려면, 음이온 denaturing 세제 나트륨 dodecyl 황산 (SDS)로 로딩 버퍼를 사용하고 5 분 95-100 ° C에서 혼합물을 끓여. 사용하기 쉽고 편리하게 로딩 버퍼 4X 샘플 버퍼입니다. </li></ol></li><li> 트레일 믹스 단백질 마커와 잘의 첫 번째 차선을로드합니다. 이 표식은 전기를 모니터링하도록 세 참조 밴드가 있습니다. 겔이 묻은 경우, 10 밴드가 10-225 KDA까지 볼 수 있습니다. </li><li> 버퍼를 실행과 함께 전기 영동 장치를 입력합니다. 페이지 젤 들어,이 트리스 - 글리신 보통 1X입니다. 자세한 버퍼 조리법은 서양 모래 바닥 페이지를 방문하십시오. EMD는 우리 OmniPur 화학 라인 고품질의 버퍼의 시약을 제공합니다. 1 시간 220V에서 젤을 실행합니다. </li><li> 단백질이 분​​리되면, 단백질이 골고루하고 균일하게 마이 그 레이션이 보장 쿠매시과 청색의 젤 얼룩. RAPIDstain가 - 사용 준비가되어있는 극도로 민감한 얼룩이다. 아무 destaining은 필요하지 않습니다. </li></ul> 3. 단백질 전송 전기 요금과 단백질은 전기 분야에서 젤을 통해 여행을 유도 할 수있는 것처럼, 그래서 단백질은 PVDF 또는 nitrocellulose 중 하나되고, 멤브레인로 젤에서 전기 분야에 전송할 수 있습니다. <ul><li> 오늘 우리는 젖은 전송을 할 것입니다. 서부 유럽 표준시 전송에서 젤 및 멤브레인는 스펀지와 종이 (스폰지 / 종이 / 젤 / 멤브레인 / 종이 / 스펀지)와 모든 기포가가 젤과 막 사이에 형성이 없습니다 확인한 후 함께 단단히 고정되어 아르 사이에 끼워 넣으면됩니다. 샌드위치는 전기장이 적용입니다 전송 버퍼에 빠져들 수 있습니다. 부정적 - 충전 단백질은 긍정적 - 충전 전극을 향해 여행을하지만, 멤브레인 그들을 중지, 그들을 바인딩, 및 계속에서 그들을 방지합니다. </li><li> 세포막의 두 종류가 있습니다 : nitrocellulose 및 PVDF. 둘 다 잘 작동. 오늘 우리는 PVDF를 사용하는 것입니다. PVDF의 점막은 methano에 몸을 담글 필요5 분 얼음 차가운 전송 버퍼로 다음 몇 분 동안 리터. 젤은 또한 얼음 차가운 전송 버퍼에서 몇 분 동안 평형을해야합니다. 이렇게하지 ​​않으면 전송 중에 젤 축소 있습니다. </li><li> 서부 유럽 표준시 전송을위한 버퍼 20 % 메탄올로 1X 트리스 - 글리신입니다. 상세 버퍼 준비는 저희 웹사이트에서 사용할 수 있습니다. </li><li> 전송이 완료되면, 아무 공기 주머니로 전송도 보장하기 위해 단백질을 시각화하는 것이 좋습니다. 이것은 쉽게 RedAlert 서양 얼룩 얼룩도 함께 수행할 수 있습니다. 이 얼룩은 - 사용 준비하고 destaining는 물로 쉽게 수행할 수 있습니다. </li></ul> 4. 항체 및 액세서리 시약 <ul><li> 항원을 탐색하고 항체를하고의 첫 번째 단계는 멤브레인을 차단하는 것입니다. 멤브레인를 차단하면 불특정 배경 바인딩을 방지할 수 있습니다. </li><li> 비 - 지방 우유 또는 BSA 중 하나는 차단 솔루션으로 사용할 수 있습니다. 그러나, phospho 특정 항체를 사용하는 경우, 그것은 그것이 높은 배경의 원인이되므로 우유를 사용하지 않는 것이 좋습니다. </li><li> EMD이 아닌 포유 동물 차단 에이전트 및 준비 얼룩 QuickBlocker입니다 SeaBlock 포함한 여러 고품질 차단 시약,,, 즉시 사용 에이전트를 차단.을 제공합니다 우리는 또한 고품질 BSA (분수 V)를 사용할 수 있습니다. <ol><li> 부드러운 교반에 따라 상온에서 막 1 시간 품어. </li><li> 부화 후 TBST 또는 PBST로 세 번 씻으십시오. TBST 또는 PBST를 만들기 위해 쉽고 편리한 방법은 (PBS 트윈의 위패 / TBS 트윈 제나) 우리의 타블렛을 사용하는 것입니다 </li></ol></li><li> 멤브레인가 차단되면, 당신은 기본 항체와 부화 준비가되어 있습니다. EMD는 30 년 이상 업계 최고의 항체 보증과 탁월한 항체의 포괄적인 포트폴리오를 제공하고 있습니다. 모든 EMD 항체와 함께, 우리는 전체 백퍼센트 노 위험 보장을 제공하는 기억하십시오. 항체를 구입하고 원하는 어떤 종류의 특이성 또는 응용 프로그램을 위해 사용합니다. 항체가 만족을 위해 노력하지 않으면, 우리는 다른 제품에 사용되는 전체 크레딧을 제공합니다. 자세한 내용은 Google 서양 모래 바닥 웹 페이지를 방문하시기 바랍니다. <ol><li> 오늘 우리는 SignalBoost의 해결 방법 1을 사용하는 우리의 기본 항체를 잠복기 것입니다. SignalBoost는 신호가 크게 향상 될 수 있도록 좋은 제품입니다. 단순히 솔루션 1 기본 항체를 품어 1 시간 동안이나 평소처럼 품어. 자세한 차단 에이전트를 추가할 필요가 없습니다. 또는 BSA 또는 차단 요원으로 무지방 건조 우유를 사용할 수 있습니다. </li><li> TBST로 씻으십시오. 우리는 사용 준비를 해산하기 위해 TBST 태블릿을 TBST합니다. </li><li> SignalBoost의 솔루션 2를 사용하여 보조 항체를 품어. 1 시간 버퍼를 차단에서 보조 항체를 품어. TBST로 다시 여러 번 멤브레인 씻으십시오. </li></ol></li></ul> 5. 검색 HRP 항체 결합 보조 들어, ECL은 전통적으로 사용됩니다. 우리가 제공하는 뛰어난 ECL 시약은 RapidStep입니다. RapidStep의 장점은 luminal 또는 확장기에 대한 혼합이 필요하지는 것입니다. 단순히 막 몇 번 스프레이하고 X - 선 필름을 사용하여 개발할 수 있습니다. RapidStep는 기판의 추가 후 낮은 그림 문 감도뿐만 아니라 최대 2 시간 동안 발광 빛을 제공합니다.

<ol>
	<li>Towbin, H., Staehelin, T., Gordon, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electrophoretic+transfer+of+proteins+from+polyacrylamide+gels+to+nitrocellulose+sheets:+procedure+and+some+applications.">Electrophoretic transfer of proteins from polyacrylamide gels to nitrocellulose sheets: procedure and some applications.</a> Proc Natl Acad Sci U S A. 76 (9), 4350-4354 (1979).</li><li>Renart, J., Reiser, J., Stark, G. R., R, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Transfer+of+proteins+from+gels+to+diazobenzyloxymethyl-paper+and+detection+with+antisera:+a+method+for+studying+antibody+specificity+and+antigen+structure.">Transfer of proteins from gels to diazobenzyloxymethyl-paper and detection with antisera: a method for studying antibody specificity and antigen structure.</a> Proc Natl Acad Sci U S A. 76 (7), 3116-3120 (1979).</li><li>Burnette, W. N. '. W. e. s. t. e. r. n. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=blotting':+electrophoretic+transfer+of+proteins+from+sodium+dodecyl+sulfate+polyacrylamide+gels+to+unmodified+nitrocellulose+and+radiographic+detection+with+antibody+and+radioiodinated+protein+A.">blotting': electrophoretic transfer of proteins from sodium dodecyl sulfate polyacrylamide gels to unmodified nitrocellulose and radiographic detection with antibody and radioiodinated protein A.</a> Analytical Biochemistry. 112, 195-203 (1981).</li><li>Ambroz, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improving+quantification+accuracy+for+Western+blots+Image+Analysis.">Improving quantification accuracy for Western blots Image Analysis.</a> , (2006).</li><li>Quan, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reactive+oxygen+species-mediated+endoplasmic+reticulum+stress+and+mitochondrial+dysfunction+contribute+to+cirsimartitin-induced+apoptosis+in+human+gallbladder+carcinoma+GBC-SD+cells.">Reactive oxygen species-mediated endoplasmic reticulum stress and mitochondrial dysfunction contribute to cirsimartitin-induced apoptosis in human gallbladder carcinoma GBC-SD cells.</a> Cancer Lett Article. , (2010).</li><li>O'Malley, D. e. r. v. i. a. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Alterations+in+colonic+corticotrophin-releasing+factor+receptors+in+the+maternally+separated+rat+model+of+irritable+bowel+syndrome:+Differential+effects+of+acute+psychological+and+physical+stressors.">Alterations in colonic corticotrophin-releasing factor receptors in the maternally separated rat model of irritable bowel syndrome: Differential effects of acute psychological and physical stressors.</a> Peptides. 31 (4), 662-670 (2010).</li></ol>

저자 안나 Eslami 제시 루얀이 문서에 사용되는 시약 및 악기를 생산 EMD 화학 병원에서 근무하고 있습니다.

이 비디오 프레 젠 테이션에서 우리는 SDS - PAGE, 항체와 프로빙 / 차단 막하고, 감지, 샘플 준비 아르 서양은 모래 바닥의 주요 단계를 강조합니다. 프로세스의 각 단계에 대해 적절한 프로토콜과 적절한 시약은 고품질 결과를 달성하는 데 사용해야합니다.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
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		<tbody>
		
<tr>
<td>CytoBuster&trade; Protein Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71009</td>
<td><a href="http://www.emdbiosciences.com/cytobuster">www.emdbiosciences.com/cytobuster</a> <a href="http://www.merck4biosciences.com/cytobuster">www.merck4biosciences.com/cytobuster</a></td>
</tr>
<tr>
<td>PhosphoSafe&trade; Extraction Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71296</td>
<td><a href="http://www.emdbiosciences.com/phosphosafe">www.emdbiosciences.com/phosphosafe</a> <a href="http://www.merck4biosciences.com/phosphosafe">www.merck4biosciences.com/phosphosafe</a></td>
</tr>
<tr>
<td>Non-Interfering Protein Assay&trade; Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>488250</td>
<td><a href="http://www.emdbiosciences.com/noninterfering">www.emdbiosciences.com/noninterfering</a> <a href="http://www.merck4biosciences.com/noninterfering">www.merck4biosciences.com/noninterfering</a></td>
</tr>
<tr>
<td>BCA Protein Assay Kit </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71285</td>
<td><a href="http://www.emdbiosciences.com/BCA">www.emdbiosciences.com/BCA</a> <a href="http://www.merck4biosciences.com/BCA">www.merck4biosciences.com/BCA</a></td>
</tr>
<tr>
<td>4X SDS Sample Buffer </td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70607</td>
<td><a href="http://www.emdbiosciences.com/4XSDS">www.emdbiosciences.com/4XSDS</a> <a href="http://www.merck4biosciences.com/4XSDS">www.merck4biosciences.com/4XSDS</a></td>
</tr>
<tr>
<td>Trail Mix&trade; Protein Markers</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>70980</td>
<td><a href="http://www.emdbiosciences.com/trailmix">www.emdbiosciences.com/trailmix</a> <a href="http://www.merck4biosciences.com/trailmix">www.merck4biosciences.com/trailmix</a></td>
</tr>
<tr>
<td>ProteoExtract&trade; Protein Extraction Kits</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/proteoextract">www.emdbiosciences.com/proteoextract</a> <a href="http://www.merck4biosciences.com/proteoextract">www.merck4biosciences.com/proteoextract</a></td>
</tr>
<tr>
<td>OmniPur&reg; Products</td>
<td>&nbsp;</td>
<td>EMD Chemicals</td>
<td>&nbsp;</td>
<td><a href="http://www.emdbiosciences.com/omnipur">www.emdbiosciences.com/omnipur</a></td>
</tr>
<tr>
<td>RAPIDstain&trade; Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>553215</td>
<td><a href="http://www.emdbiosciences.com/rapidstain">www.emdbiosciences.com/rapidstain</a> <a href="http://www.merck4biosciences.com/rapidstain">www.merck4biosciences.com/rapidstain</a></td>
</tr>
<tr>
<td>RedAlert&trade; 10X Western Blot Stain</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>71078</td>
<td><a href="http://www.emdbiosciences.com/redalert">www.emdbiosciences.com/redalert</a> <a href="http://www.merck4biosciences.com/redalert">www.merck4biosciences.com/redalert</a></td>
</tr>
<tr>
<td>SeaBlock&trade; Reagent, Salmon Plasma</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>558300</td>
<td><a href="http://www.emdbiosciences.com/seablock">www.emdbiosciences.com/seablock</a> <a href="http://www.merck4biosciences.com/seablock">www.merck4biosciences.com/seablock</a></td>
</tr>
<tr>
<td>BLOT-QuickBlocker Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>WB57</td>
<td><a href="http://www.emdbiosciences.com/quickblocker">www.emdbiosciences.com/quickblocker</a> <a href="http://www.merck4biosciences.com/quickblocker">www.merck4biosciences.com/quickblocker</a></td>
</tr>
<tr>
<td>Albumin, Bovine Serum, Fraction V</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>12660</td>
<td><a href="http://www.emdbiosciences.com/bovineserum">www.emdbiosciences.com/bovineserum</a> <a href="http://www.merck4biosciences.com/bovineserum">www.merck4biosciences.com/bovineserum</a></td>
</tr>
<tr>
<td>PBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524653</td>
<td><a href="http://www.emdbiosciences.com/PBST">www.emdbiosciences.com/PBST</a> <a href="http://www.merck4biosciences.com/PBST">www.merck4biosciences.com/PBST</a></td>
</tr>
<tr>
<td>TBS-TWEEN&reg; Tablets</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>524753</td>
<td><a href="http://www.emdbiosciences.com/TBST">www.emdbiosciences.com/TBST</a> <a href="http://www.merck4biosciences.com/TBST">www.merck4biosciences.com/TBST</a></td>
</tr>
<tr>
<td>SignalBoost&trade; Immunoreaction Enhancer Kit</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>407207</td>
<td><a href="http://www.emdbiosciences.com/signalboost">www.emdbiosciences.com/signalboost</a> <a href="http://www.merck4biosciences.com/signalboost">www.merck4biosciences.com/signalboost</a></td>
</tr>
<tr>
<td>RapidStep&trade; ECL Reagent</td>
<td>&nbsp;</td>
<td>EMD Chemicals/Merck KGaA</td>
<td>345818</td>
<td><a href="http://www.emdbiosciences.com/rapidstep">www.emdbiosciences.com/rapidstep</a> <a href="http://www.merck4biosciences.com/rapidstep">www.merck4biosciences.com/rapidstep</a></td>
</tr>		</tbody>
		</table>
		</div>

western blotting: sample preparation to detection

모래 바닥 웨스턴 조직 homogenate 또는 추출 주어진 샘플에서 특정 단백질을 감지하는 데 사용되는 분석 기술입니다. 그것은 polypeptide의 길이 (denaturing 조건) 또는 단백질의 3 차원 구조 (기본 / 비 denaturing 조건)에 의해 기본 또는 변성 단백질을 분리하는 겔 전기 영동을 사용합니다. 단백질은 다음 대상 단백질에 특정한 항체를 사용하여 그들이 탐지하는 막 (일반적으로 nitrocellulose 또는 PVDF), (감지)으로 전송됩니다.

Watch this Scientific Journal Video about Western Blotting: Sample Preparation to Detection at JoVE.com

Western Blotting: Sample Preparation to Detection

모래 바닥 웨스턴 조직 homogenate 또는 추출 주어진 샘플에서 특정 단백질을 감지하는 데 사용되는 분석 기술입니다.

웨스턴 모래 바닥 : 샘플 준비를 탐지하기 위해

Western blotting is an analytical technique used to detect specific proteins in a given sample of tissue homogenate or extract. It uses gel ...

western-blotting-sample-preparation-to-detection

Research

JoVE Journal

Biology

138.9K 조회수.  EMD Chemicals Inc.. 모래 바닥 웨스턴 조직 homogenate 또는 추출 주어진 샘플에서 특정 단백질을 감지하는 데 사용되는 분석 기술입니다.

비디오: Western Blotting: Sample Preparation to Detection

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

든 샘플 준비 : 울트라 씬 레이어 방법

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

연구

생물학

Western Blotting Using the Invitrogen NuPage Novex Bis Tris MiniGels

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the relative amount of the protein present in different samples, and analyze the results of co-immunoprecipitation experiments. In this method, a target protein is detected with a specific primary antibody in a given sample of tissue homogenate or extract. Protein separation according to molecular weight is achieved using denaturing SDS-PAGE. After transfer to a membrane, the target protein is probed with a specific primary antibody and detected by chemiluminescence.
Since its first description, the western-blotting technique has undergone several improvements, including pre-cast gels and user-friendly equipment. In our laboratory, we have chosen to use the commercially available NuPAGE electrophoresis system from Invitrogen. It is an innovative neutral pH, discontinuous SDS-PAGE, pre-cast mini-gel system. This system presents several advantages over the traditional Laemmli technique including: i) a longer shelf life of the pre-cast gels ranging from 8 months to 1 year; ii) a broad separation range of molecular weights from 1 to 400 kDa depending of the type of gel used; and iii) greater versatility (range of acrylamide percentage, the type of gel, and the ionic composition of the running buffer).
The procedure described in this video article utilizes the Bis-Tris discontinuous buffer system with 4-12% Bis-Tris gradient gels and MES running buffer, as an illustration of how to perform a western-blot using the Invitrogen NuPAGE electrophoresis system. In our laboratory, we have obtained good and reproducible results for various biochemical applications using this western-blotting method.

This technical article describes a standard western-blotting procedure using the commercially available NuPAGE electrophoresis Mini-Gel system from Invitrogen.

웨스턴 Invitrogen NuPage Novex BIS 트리스 MiniGels를 사용하여 모래 바닥

Western Blotting (or immunoblotting) is a standard laboratory procedure allowing investigators to verify the expression of a protein, determine the ...

Proteomic Sample Preparation from Formalin Fixed and Paraffin Embedded Tissue

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale quantitative analysis of formalin fixed and paraffin embedded (FFPE) tissue. The procedure comprises four distinct steps. The first one is the preparation of sections from the FFPE material and microdissection of cells of interest. In the second step the isolated cells are lysed and processed using 'filter aided sample preparation' (FASP) technique. In this step, proteins are depleted from reagents used for the sample lysis and are digested in two-steps using endoproteinase LysC and trypsin. 
After each digestion, the peptides are collected in separate fractions and their content is determined using a highly sensitive fluorescence measurement. Finally, the peptides are fractionated on 'pipette-tip' microcolumns. The LysC-peptides are separated into 4 fractions whereas the tryptic peptides are separated into 2 fractions. In this way prepared samples allow analysis of proteomes from minute amounts of material to a depth of 10,000 proteins. Thus, the described workflow is a powerful technique for studying diseases in a system-wide-fashion as well as for identification of potential biomarkers and drug targets.

Archival formalin fixed and paraffin embedded (FFPE) clinical samples are valuable material for investigation of diseases. Here we demonstrate a sample preparation workflow allowing in-depth proteomic analysis of microdissected FFPE tissue.

포르말린에서 프로테오믹스 샘플 준비 파라핀 조직에게 고정 및

Preserved clinical material is a unique source for proteomic investigation of human disorders. Here we describe an optimized protocol allowing large scale ...

V3 Stain-free Workflow for a Practical, Convenient, and Reliable Total Protein Loading Control in Western Blotting

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black box&#34; because an experimentalist does not know if it has been performed successfully until the last of several steps. Moreover, the quality of western blot data is sometimes challenged due to a lack of effective quality control tools in place throughout the western blotting process. Here we describe the V3 western workflow, which applies stain-free technology to address the major concerns associated with the traditional western blot protocol. This workflow allows researchers: 1) to run a gel in about 20-30 min; 2) to visualize sample separation quality within 5 min after the gel run; 3) to transfer proteins in 3-10 min; 4) to verify transfer efficiency quantitatively; and most importantly 5) to validate changes in the level of the protein of interest using total protein loading control. This novel approach eliminates the need of stripping and reprobing the blot for housekeeping proteins such as &#946;-actin, &#946;-tubulin, GAPDH, etc. The V3 stain-free workflow makes the western blot process faster, transparent, more quantitative&#160;and reliable.

V3 workflow is a western blot procedure using stain-free gels. The stain-free technology allows researchers to visualize protein separation quality, to verify the transfer efficiency, and most importantly, to validate the change in the protein of interest using total protein quantification as a reliable loading control.

서부 블로팅에서 실용적이고 편리하며 신뢰할 수 있는 토탈 단백질 로딩 제어를 위한 V3 얼룩 없는 워크플로우

The western blot is a very useful and widely adopted lab technique, but its execution is challenging. The workflow is often characterized as a &#34;black ...

The Fastest Western in Town: A Contemporary Twist on the Classic Western Blot Analysis

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of Western blotting has several drawbacks that include low quality resolution, spurious bands, decreased sensitivity, and poor protein integrity. Recent advances have drastically improved numerous aspects of the standard Western blot protocol to produce higher qualitative and quantitative data. The Bis-Tris gel system, an alternative to the conventional Laemmli system, generates better protein separation and resolution, maintains protein integrity, and reduces electrophoresis to a 35 min run time. Moreover, the iBlot dry blotting system, dramatically improves the efficacy and speed of protein transfer to the membrane in 7 min, which is in contrast to the traditional protein transfer methods that are often more inefficient with lengthy transfer times. In combination with these highly innovative modifications, protein detection using infrared fluorescent imaging results in higher-quality, more accurate and consistent data compared to the standard Western blotting technique of chemiluminescence. This technology can simultaneously detect two different antigens on the same membrane by utilizing two-color near-infrared dyes that are visualized in different fluorescent channels. Furthermore, the linearity and broad dynamic range of fluorescent imaging allows for the precise quantification of both strong and weak protein bands. Thus, this protocol describes the key improvements to the classic Western blotting method, in which these advancements significantly increase the quality of data while greatly reducing the performance time of this experiment.

This protocol explores the latest advancements in performing Western blot analyses.&#160;These novel modifications employ a Bis-Tris gel system with a 35&#160;min electrophoresis run time, a 7&#160;min dry blotting transfer system, and infrared fluorescent protein detection and imaging that generates higher resolution, quality, sensitivity, and improved accuracy of Western data.

타운 웨스턴 가장 빠른 : 클래식 웨스턴 블롯 분석에 현대 트위스트

The Western blot techniques that were originally established in the late 1970s are still actively utilized today. However, this traditional method of ...

A Guide to Modern Quantitative Fluorescent Western Blotting with Troubleshooting Strategies

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins within complex biological samples. Since then, western blotting methodology has become a common component of the molecular biologists experimental repertoire. A cursory search of PubMed using the term &#8220;western blot&#8221; suggests that in excess of two hundred and twenty thousand published manuscripts have made use of this technique by the year 2014. Importantly, the last ten years have seen technical imaging advances coupled with the development of sensitive fluorescent labels which have improved sensitivity and yielded even greater ranges of linear detection. The result is a now truly Quantifiable Fluorescence based Western Blot (QFWB) that allows biologists to carry out comparative expression analysis with greater sensitivity and accuracy than ever before. Many &#8220;optimized&#8221; western blotting methodologies exist and are utilized in different laboratories. These often prove difficult to implement due to the requirement of subtle but undocumented procedural amendments. This protocol provides a comprehensive description of an established and robust QFWB method, complete with troubleshooting strategies.

The advancement of western blotting using fluorescence has allowed detection of subtle changes in protein expression enabling quantitative analyses. Here we describe a robust methodology for detection of a range of proteins across a variety of species and tissue types. A strategy to overcome common technical problems is also provided.

문제 해결 전략 현대 양적 형광 웨스턴 블로 팅에 가이드

The late 1970s saw the first publicly reported use of the western blot, a technique for assessing the presence and relative abundance of specific proteins ...

Mucin Agarose Gel Electrophoresis: Western Blotting for High-molecular-weight Glycoproteins

Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against invading pathogens. The main role of these macromolecules is to facilitate particle trapping and clearance while promoting lubrication of the mucosa. During protein synthesis, mucins undergo intense O-glycosylation and multimerization, which dramatically increase the mass and size of these molecules. These post-translational modifications are critical for the viscoelastic properties of mucus. As a result of the complex biochemical and biophysical nature of these molecules, working with mucins provides many challenges that cannot be overcome by conventional protein analysis methods. For instance, their high-molecular-weight prevents electrophoretic migration via regular polyacrylamide gels and their sticky nature causes adhesion to experimental tubing. However, investigating the role of mucins in health (e.g., maintaining mucosal integrity) and disease (e.g., hyperconcentration, mucostasis, cancer) has recently gained interest and mucins are being investigated as a therapeutic target. A better understanding of the production and function of mucin macromolecules may lead to novel pharmaceutical approaches, e.g., inhibitors of mucin granule exocytosis and/or mucolytic agents. Therefore, consistent and reliable protocols to investigate mucin biology are critical for scientific advancement. Here, we describe conventional methods to separate mucin macromolecules by electrophoresis using an agarose gel, transfer protein into nitrocellulose membrane, and detect signal with mucin-specific antibodies as well as infrared fluorescent gel reader. These techniques are widely applicable to determine mucin quantitation, multimerization and to test the effects of pharmacological compounds on mucins.

Mucins are high-molecular-weight glycoconjugates, with size ranging from 0.2 to 200 megadalton (MDa). As a result of their size, mucins do not penetrate conventional polyacrylamide gels and require larger pores for separation. We provide a detailed protocol for mucin agarose gel electrophoresis to assess relative quantification and study polymer assembly.

점액 아가로 오스 겔 전기 영동 : 높은 분자량 당 단백질에 대한 웨스턴 블로 팅


Mucins, the heavily-glycosylated proteins lining mucosal surfaces, have evolved as a key component of innate defense by protecting the epithelium against ...

Sample Preparation for Mass Cytometry Analysis

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities for deep analysis well beyond what is possible with conventional fluorescence-based flow cytometry. As with any new technology, there are critical steps that help ensure the reliable generation of high-quality data. Presented here is an optimized protocol that incorporates multiple techniques for the processing of cell samples for mass cytometry analysis. The methods described here will help the user avoid common pitfalls and achieve consistent results by minimizing variability, which can lead to inaccurate data. To inform experimental design, the rationale behind optional or alternative steps in the protocol and their efficacy in uncovering new findings in the biology of the system being investigated is covered. Lastly, representative data is presented to illustrate expected results from the techniques presented here.

This article describes the collection and processing of samples for mass cytometry analysis.

대량 세포 계측법 분석을위한 샘플 준비

Mass cytometry utilizes antibodies conjugated with heavy metal labels, an approach that has greatly increased the number of parameters and opportunities ...

Sample Preparation and Imaging of Exosomes by Transmission Electron Microscopy

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The contents and morphology of the secreted vesicles reflect cell behavior or physiological status, for example cell growth, migration, cleavage, and death. The exosomes&#39; role may depend highly on size, and the size of exosomes varies from 30 to 300 nm. The most widely used method for exosome imaging is negative staining, while other results are based on Cryo-Transmission Electron Microscopy, Scanning Electron Microscopy, and Atomic Force Microscopy. The typical exosome&#39;s morphology assessed through negative staining is a cup-shape, but further details are not yet clear. An exosome well-characterized through structural study is necessary particular in medical and pharmaceutical fields. Therefore, function-dependent morphology should be verified by electron microscopy techniques such as labeling a specific protein in the detailed structure of exosome. To observe detailed structure, ultrathin sectioned images and negative stained images of exosomes were compared. In this protocol, we suggest transmission electron microscopy for the imaging of exosomes including negative staining, whole mount immuno-staining, block preparation, thin section, and immuno-gold labelling.

This protocol describes the various techniques necessary for transmission electron microscopy including negative staining, ultrathin sectioning for detailed structure, and immuno-gold labelling to determine the positions of specific proteins in exosomes.

샘플 준비 및 전송 전자 현미경 검사 법에 의해 Exosomes의 영상

Exosomes are nano-sized extracellular vesicles secreted by body fluids and are known to represent the characteristics of cells that secrete them. The ...

Miniaturized Sample Preparation for Transmission Electron Microscopy

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein complexes to atomic resolution. However, protein isolation techniques and sample preparation methods for EM remain a bottleneck. A relatively small number (100,000 to a few million) of individual protein particles need to be imaged for the high-resolution analysis of proteins by the single particle EM approach, making miniaturized sample handling techniques and microfluidic principles feasible.
A miniaturized, paper-blotting-free EM grid preparation method for sample pre-conditioning, EM grid priming and post processing that only consumes nanoliter-volumes of sample is presented. The method uses a dispensing system with sub-nanoliter precision to control liquid uptake and EM grid priming, a platform to control the grid temperature thereby determining the relative humidity above the EM grid, and a pick-and-plunge-mechanism for sample vitrification. For cryo-EM, an EM grid is placed on the temperature-controlled stage and the sample is aspirated into a capillary. The capillary tip is positioned in proximity to the grid surface, the grid is loaded with the sample and excess is re-aspirated into the microcapillary. Subsequently, the sample film is stabilized and slightly thinned by controlled water evaporation regulated by the offset of the platform temperature relative to the dew-point. At a given point the pick-and-plunge mechanism is triggered, rapidly transferring the primed EM grid into liquid ethane for sample vitrification. Alternatively, sample-conditioning methods are available to prepare nanoliter-sized sample volumes for negative stain (NS) EM.
The methodologies greatly reduce sample consumption and avoid approaches potentially harmful to proteins, such as the filter paper blotting used in conventional methods. Furthermore, the minuscule amount of sample required allows novel experimental strategies, such as fast sample conditioning, combination with single-cell lysis for &#34;visual proteomics,&#34; or &#34;lossless&#34; total sample preparation for quantitative analysis of complex samples.

An instrument and methods for the preparation of nanoliter-sized sample volumes for transmission electron microscopy is presented. No paper-blotting steps are required, thus avoiding the detrimental consequences this can have for proteins, significantly reducing sample loss and enabling the analysis of single cell lysate for visual proteomics.

전송 전자 현미경 검사 법에 대 한 소형된 샘플 준비

Due to recent technological progress, cryo-electron microscopy (cryo-EM) is rapidly becoming a standard method for the structural analysis of protein ...