1. تحضير العينة الخطوة الأولى في غرب النشاف هو إعداد العينة. لإعداد العينات لتشغيل الخلايا والأنسجة هلام يلزم lysed للافراج عن بروتينات الفائدة. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> ومريحة ، والجاهزة للاستخدام كاشف التي هي متعددة وسهلة الاستخدام لاستخراج البروتين غير قابلة للذوبان أو CytoBuster PhosphoSafe. تحتوي هذه المخازن استخراج المنظفات الأمثل لكفاءة الاستخلاص من البروتينات القابلة للذوبان من خلايا الثدييات والحشرات. لطيف ، غير الأيونية تكوين البروتين CytoBuster الكاشف استخراج تمكن من العزلة الذاتية الفعالة وظيفيا أو البروتينات وأعرب دون الحاجة إلى المعالجة الثانوية مثل صوتنة أو تجميد / الذوبان. PhoshoSafe له فائدة اضافية لاحتواء مثبطات الفوسفاتيز الأربعة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بيليه الخلايا بواسطة الطرد المركزي سرعة منخفضة (على سبيل المثال 5 دقائق في 2500 XG) ، بيليه الخلية استنزاف جيدا. </li><li style=";text-align:right;direction:rtl"> Resuspend الخلايا في البروتين CytoBuster باستخدام الكاشف استخراج 150 ميكرولتر في الخلايا 6 10 (المبلغ الأمثل للبروتين CytoBuster الكاشف استخراج قد تختلف استنادا إلى حجم الخلية). </li><li style=";text-align:right;direction:rtl"> يحضن في درجة حرارة الغرفة لمدة 5 دقائق. </li><li style=";text-align:right;direction:rtl"> نقل إلى أنبوب مناسبة وتدور لمدة 5 دقائق في XG 16000 في 4 درجات مئوية. </li><li style=";text-align:right;direction:rtl"> مسح نقل طاف (استخراج الخلايا) إلى أنبوب جديد والمضي قدما في التحليل. </li></ol></li><li style=";text-align:right;direction:rtl"> لاستخراج البروتين المتخصصة ، ونحن نوصي باستخدام لدينا عالية الجودة أطقم ProteoExtract. EMD ما يزيد على اثني عشر مجموعات لعزل الميتوكوندريا ، التحت خلوية استخراج بروتيوم ، عبر الغشاء استخراج البروتين ، وغيرها الكثير. يرجى زيارتنا لطخة غربية الصفحة المقصودة لمزيد من التفاصيل. </li><li style=";text-align:right;direction:rtl"> EMD تبيع أيضا تشكيلة واسعة من مجموعات البروتيني والفوسفاتيز كوكتيل المانع. حالما يحدث تحلل ، التحلل البروتيني ، ونزع الفسفات تمسخ تبدأ. يمكن أن تكون هذه الأحداث باستمرار تباطأ بشكل كبير إذا ما حافظت على عينات من الجليد أو عند 4 درجة مئوية تضاف في كل الأوقات ومثبطات مناسبة جديدة للتحلل العازلة. يرجى زيارتنا لطخة غربية الصفحة المقصودة لمزيد من التفاصيل. </li></ul> 2. SDS - PAGE الخطوة الثانية في غرب النشاف هو فصل البروتين. يتم فصل البروتينات على أساس الوزن الجزيئي. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> يمكنك جعل الصفحة الخاصة بك هلام ، ولكن سنكون باستخدام تجاريا مسبقة الصنع هلام. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> بعد إعداد نموذج الخاص بك ، كنت على استعداد لتحديد تركيز البروتين. EMD ومجموعتين لهذا لقياس التركيز ، واحد يجري الفحص البروتين عدم التدخل كيت والآخر يجري الفحص البروتين BCA كيت. </li><li style=";text-align:right;direction:rtl"> حالما يتم تقييم تركيز البروتين نحن مستعدون لإعداد نموذج لدينا لتحميل الجل. الأضداد تعترف عادة جزء صغير من البروتين من الفائدة (ويشار إليه حاتمة) وهذا المجال قد تتواجد داخل التشكل 3D من البروتين. لتمكين وصول الأجسام المضادة لهذا الجزء من الضروري أن تتكشف البروتين. </li><li style=";text-align:right;direction:rtl"> لتفسد ، واستخدام العازلة تحميل مع أنيوني كبريتات الصوديوم المنظفات تغيير طبيعة دوديسيل (SDS) ، ويغلي الخليط في 95-100 درجة مئوية لمدة 5 دقائق. وجود مخزن مؤقت التحميل سهلة ومريحة لاستخدام الاحتياطي 4X هو نموذج. </li></ol></li><li style=";text-align:right;direction:rtl"> تحميل المسار الأول من البئر مع تريل ميكس ماركر البروتين. وهذه العلامة المرجع العصابات الثلاث التي تسمح لك لرصد الكهربائي. عندما اتسخت الجل ، والعصابات 10 مرئية تتراوح 10-225 كيلو دالتون. </li><li style=";text-align:right;direction:rtl"> سد الوحدة الكهربائي مع تشغيل العازلة. لPAGE المواد الهلامية ، وهذا هو عادة تريس 1X - جليكاين. العازلة عن وصفات مفصلة ، يمكنك زيارة صفحة الغربية النشاف. EMD عروض عالية الجودة مخازن الكواشف الكيماوية مع خطنا OmniPur. تشغيل في هلام 220V لمدة 1 ساعة. </li><li style=";text-align:right;direction:rtl"> مرة واحدة في البروتينات وفصلها ، وصمة عار مع الجل الأزرق Coomassie لضمان البروتينات هاجروا بالتساوي وبشكل موحد. RAPIDstain هو وصمة عار فائقة الحساسية التي هي جاهزة للاستخدام. ليس هناك حاجة destaining. </li></ul> 3. نقل البروتين تماما كما يمكن أن يتسبب البروتينات مع شحنة كهربائية من خلال السفر إلى هلام في حقل الكهرباء ، بحيث يمكن نقل البروتينات في مجال كهربائي من الجل على غشاء ، ويجري إما PVDF أو النيتروسليلوز. <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اليوم سوف نقوم بنقل الرطب. في مجال نقل الرطب ، وتقع الجل والغشاء بين الإسفنج ورقة (الاسفنج / ورقة / هلام / غشاء / ورقة / الاسفنج) ، وفرضت جميع بإحكام معا بعد التأكد من أي فقاعات هواء شكلت بين جل والأغشية. الغارقة في شطيرة في نقل العازلة التي يتم تطبيق حقل كهربائي. البروتينات المشحونة سلبا السفر نحو القطب المشحون إيجابيا ، ولكن توقف عن غشاء لهم ، يربط بينها ، ويمنعها من الاستمرار في. </li><li style=";text-align:right;direction:rtl"> نوعان من الأغشية متوفرة : النيتروسليلوز وPVDF. تعمل بشكل جيد على حد سواء. اليوم سنكون باستخدام PVDF. الأغشية PVDF تتطلب تمرغ في methanoل لبضع دقائق ، ثم نقل العازلة الجليد البارد لمدة 5 دقائق. الجل يحتاج أيضا للتوازن لبضع دقائق في نقل العازلة الجليد الباردة. قد لا تفعل ذلك تقليص هلام أثناء النقل. </li><li style=";text-align:right;direction:rtl"> المخزن المؤقت لنقل الرطب 1X تريس - جليكاين مع الميثانول 20 ٪. استعدادات مفصلة العازلة متاحة على موقعنا على الانترنت. </li><li style=";text-align:right;direction:rtl"> بمجرد اكتمال عملية النقل ، انها فكرة جيدة لرؤية البروتينات لضمان نقل حتى مع عدم وجود جيوب الهواء. ويمكن بسهولة أن يتم ذلك مع RedAlert الغربية وصمة عار وصمة عار. هذه وصمة عار على استعداد للاستخدام ويمكن القيام به بسهولة مع destaining المياه. </li></ul> 4. الأجسام المضادة والكواشف ملحقة <ul style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> الخطوة الأولى في التحقيق والقيام على الضد مستضد هو كتلة الغشاء. حظر يمنع الغشاء غير محددة وملزمة الخلفية. </li><li style=";text-align:right;direction:rtl"> ويمكن استخدام إما منزوع الدسم أو اللبن BSA كحل حظر. ومع ذلك ، عند استخدام الفوسفات محددة الأجسام المضادة ، فمن غير المستحسن استخدام الحليب لأنه سيتسبب في ارتفاع الخلفية. </li><li style=";text-align:right;direction:rtl"> EMD عدة عروض عالية الجودة الكواشف حظر ، بما في ذلك SeaBlock ، وهو عامل غير الثدييات الحجب وQuickBlocker BLOT ، الذي يعد وجاهزة للاستخدام عامل محصر. لدينا أيضا عالية الجودة BSA (V الكسر) متوفرة. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> احتضان الغشاء 1 ساعة في درجة حرارة الغرفة في إطار التحريض لطيف. </li><li style=";text-align:right;direction:rtl"> يغسل ثلاث مرات في TBST أو PBST بعد الحضانة. طريقة سهلة ومريحة لجعل TBST أو PBST هو استخدام أقراص لدينا (PBS توين أقراص / أقراص TBS توين) </li></ol></li><li style=";text-align:right;direction:rtl"> مرة واحدة وقد تم حظر الغشاء ، كنت على استعداد لاحتضان مع الأجسام المضادة الأولية. وقد تقدم EMD مجموعة شاملة من الأضداد استثنائية ، وأفضل ضمانة للأجسام المضادة في هذه الصناعة لأكثر من 30 عاما. تذكر أن مع كل الأضداد EMD ، ونحن نقدم كامل 100 ٪ لا يوجد ضمان للخطر. شراء الأضداد واستخدامها لأية خصوصية الأنواع أو التطبيق الذي يرغبه. إذا كان الضد لا يعمل بشكل يرضيك ، فإننا سوف توفر الائتمان الكامل ليتم استخدامه على أي منتج آخر. لمزيد من التفاصيل ، يرجى زيارة صفحة الويب النشاف الغربية. <ol style=";text-align:right;direction:rtl"><li style=";text-align:right;direction:rtl"> اليوم سنكون احتضان الضد لدينا الأولية باستخدام حل 1 من SignalBoost. SignalBoost هو منتج كبير السماح تعزيز إشارة إلى حد كبير. ببساطة احتضان الضد الخاص الأساسي في الحل (1) ويحضن لمدة 1 ساعة أو كما تفعل عادة. ليست هناك حاجة لإضافة المزيد من عوامل عرقلة. بدلا من ذلك ، يمكنك استخدام جيش صرب البوسنة أو غير دهن الحليب الجاف كعامل حظر. </li><li style=";text-align:right;direction:rtl"> يغسل مع TBST. نجعل TBST باستخدام - الجاهزة للقرص TBST حل. </li><li style=";text-align:right;direction:rtl"> احتضان الضد الخاص به حل الثانوية 2 من SignalBoost. احتضان الجسم المضاد الثانوي في حجب العازلة لمدة 1 ساعة. يغسل الغشاء مرة أخرى عدة مرات مع TBST. </li></ol></li></ul> 5. كشف HRP عن الأجسام المضادة الثانوية التصريف ، يستخدم عادة ECL. وكاشف ممتازة ECL نقدمها هي RapidStep. مزايا RapidStep هي أنه لا يلزم من الاختلاط أو محسن اللمعية. رذاذ ببساطة الغشاء بضع مرات ، وتطوير استخدام الأشعة السينية الفيلم. RapidStep عروض حساسية منخفضة فضلا عن الرسم التخطيطي للضوء ينبعث منها لمدة تصل إلى 2 ساعة بعد إضافة الركيزة.

<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>

ويعمل في كتاب اسلامي وآنا لوجان جيسي EMD من قبل شركة كيماويات التي تنتج الكواشف والأدوات المستخدمة في هذه المادة.

في هذا العرض الفيديو ، فقد أبرزنا الخطوات الرئيسية في غرب النشاف التي هي إعداد العينات ، SDS - PAGE ، غشاء حجب / التحقيق مع الأجسام المضادة ، والكشف. لكل خطوة من العملية ، يجب استخدام البروتوكولات المناسبة والكواشف المناسبة لتحقيق نتائج عالية الجودة.

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		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<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

النشاف الغربي هو أسلوب التحليل المستخدمة للكشف عن بروتينات معينة في عينة معينة من جناسة الأنسجة أو استخراج. ويستخدم الكهربائي للهلام لفصل البروتينات الأصلي أو التشويه والتحريف من طول ببتيد (شروط تغيير طبيعة) أو عن طريق هيكل 3 - D من البروتين (أصلي / عدم تغيير طبيعة الظروف). ثم يتم نقل البروتينات إلى غشاء (عادة النيتروسليلوز أو PVDF) ، حيث يتم بحث (الكشف) باستخدام أجسام مضادة محددة للبروتين الهدف.

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

Western Blotting: Sample Preparation to Detection

النشاف الغربي هو أسلوب التحليل المستخدمة للكشف عن بروتينات معينة في عينة معينة من جناسة الأنسجة أو استخراج.

الغربية التنشيف : إعداد نموذج لكشف

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

139.0K Views.  EMD Chemicals Inc.. النشاف الغربي هو أسلوب التحليل المستخدمة للكشف عن بروتينات معينة في عينة معينة من جناسة الأنسجة أو استخراج.

Video: 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).

MALDI تحضير العينة : الترا أسلوب طبقة رقيقة

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

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.

الغربية التنشيف باستخدام NuPage Invitrogen Novex مكرر تريس MiniGels

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.

إعداد نموذج البروتين من الفورمالين الثابتة والبارافين جزءا لا يتجزأ من نسيج

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.

V3 سير العمل خالية من وصمة عار لمراقبة عملية ومريحة وموثوق بها مجموع البروتين تحميل في النشاف الغربية

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.

أسرع الغربية في المدينة: لمسة عصرية على تحليل كلاسيكي غربي وصمة عار

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.

دليل لمودرن الكمية نيون الغربية التنشيف مع استراتيجيات استكشاف الأخطاء وإصلاحها

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.

الميوسين الاغاروز جل الكهربائي: ويسترن التنشيف للبروتينات سكرية عالية الوزن الجزيئي


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.

إعداد نموذج لتحليل قداس الخلوي

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.

نموذج إعداد وتصوير اكسوسوميس مجهر إلكتروني

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.

إعداد عينة المنمنمة مجهر إلكتروني

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