<ol>
	<li>Christensen, L. L. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Theoretical+analysis+of+protein+concentration+determination+using+biosensor+technology+under+conditions+of+partial+mass+transport+limitation.">Theoretical analysis of protein concentration determination using biosensor technology under conditions of partial mass transport limitation.</a> Anal. Biochem. 249, 153-164 (1997).</li><li>Sigmundsson, K., M&#226;sson, G., Rice, R., Beauchemin, N., &#214;brink, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Determination+of+active+concentrations+and+association+and+dissociation+rate+constants+of+interacting+biomolecules:+an+analytical+solution+to+the+theory+for+kinetic+and+mass+transport+limitations+in+biosensor+technology+and+its+experimental+verification.">Determination of active concentrations and association and dissociation rate constants of interacting biomolecules: an analytical solution to the theory for kinetic and mass transport limitations in biosensor technology and its experimental verification.</a> Biochemistry. 41 (26), 8263-8276 (2002).</li><li>Stubbs, M. T., Laber, B., Bode, W., Huber, R., Jerala, R., Lenarcic, B., Turk, V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+refined+2.4+A+X-ray+crystal+structure+of+recombinant+human+stefin+B+in+complex+with+the+cysteine+proteinase+papain:+a+novel+type+of+proteinase+inhibitor+interaction.">The refined 2.4 A X-ray crystal structure of recombinant human stefin B in complex with the cysteine proteinase papain: a novel type of proteinase inhibitor interaction.</a> EMBO J. 9 (6), 1939-1947 (1990).</li><li>Pol, E., Bj&#246;rk, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Importance+of+the+second+binding+loop+and+the+C-terminal+end+of+cystatin+B+(Stefin+B)+for+inhibition+of+cysteine+proteinases.">Importance of the second binding loop and the C-terminal end of cystatin B (Stefin B) for inhibition of cysteine proteinases.</a> Biochemistry. 38 (32), 10519-10526 (1999).</li><li>Bj&#246;rk, I., Pol, E., Raub-Segall, E., Abrahamson, M., Rowan, A. D., Mort, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Differential+changes+in+the+association+and+dissociation+rate+constants+for+binding+of+cystatins+to+target+proteinases+occurring+on+N-terminal+truncation+of+the+inhibitors+indicate+that+the+interaction+mechanism+varies+with+different+enzymes.">Differential changes in the association and dissociation rate constants for binding of cystatins to target proteinases occurring on N-terminal truncation of the inhibitors indicate that the interaction mechanism varies with different enzymes.</a> Biochem. J. 299, 219-225 (1994).</li><li>Pace, C. N., Vajdos, F., Fee, L., Grimsley, G., Gray, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=How+to+measure+and+predict+the+molar+absorption+coefficient+of+a+protein.">How to measure and predict the molar absorption coefficient of a protein.</a> ProteinScience. 4 (11), 2411-2423 (1995).</li></ol>

يعمل مقدم البلاغ للرعاية الصحية GE التي تنتج الكواشف والأدوات المستخدمة في هذه المقالة.

في هذا العمل ، وأنتج أربعة المسوخ البرية وباء cystatin نوع من أجل تقييم أهمية حلقة الربط الثاني وتنتهي C - محطة للتفاعل بين B cystatin وغراء. أظهرت هذه الدراسة الميزة وسهولة استخدام X100 Biacore لتحديد تركيز محددة ملزمة ، وتحليل حركية من البروتين البروتين التفاعلات من أجل فهم بنية وظيفة العلاقات. أثبتنا أن مجموع قياس تركيز البروتين لا تكشف المتغيرات البروتين التي خفضت النشاط ملزم ، وهو في هذه الحالة يقدم الخطأ قياس كبير في تحديد تقارب ملزمة وحركية. وتحددت تركيزات محددة وملزمة من المتغيرات B cystatin باستخدام CFCA مع X100 Biacore. أدى تركيز باستخدام القياسات التي CFCA كمدخل لتحليل الحركية في معدل موثوق بها والثوابت تقارب ، مما يسمح للالتفسير الصحيح لآلية التفاعل. كانت الانتماءات انخفض من المسوخ على وجه الحصر تقريبا بسبب القيمة د ك زيادة ، في حين كان ك تتأثر إلا قليلا. هذا السلوك يشير إلى أن كلا من المنطقة الثانية حلقة الربط ونهاية C - المحطة ليست مهمة بالنسبة لمعدل ربط المانع للغراء. بدلا من ذلك ، أنها تسهم في تقارب ملزمة في المقام الأول عن طريق الحفاظ على مثبطات الانزيم تعلق على مرة واحدة وقد تم تشكيل المجمع.

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		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>Biacore&trade; X100 System</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1100-73</td>
<td><a href="http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html">http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html</a></td>
</tr>
<tr>
<td>Biacore&trade; X100 Plus Package</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1007-98</td>
<td><a href="http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html">http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html</a></td>
</tr>
<tr>
<td>Sensor Chip CM5</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1000-14</td>
<td><a href="http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html">http://www.biacore.com/lifesciences/products/systems_overview/x100/system_information/index.html</a></td>
</tr>
<tr>
<td>Amine Coupling Kit</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1000-50</td>
<td>&nbsp;</td>
<tr>
<td>Acetate buffer pH 4.5, 50 ml</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1003-50</td>
<td>&nbsp;</td>
<tr>
<td>HBS-EP+ buffer 10X, 4 x 50 ml</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1008-26</td>
<td>&nbsp;</td>
<tr>
<td>Plastic Vials &Oslash; 11 mm</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1002-87</td>
<td>&nbsp;</td>
<tr>
<td>Rubber caps, type 2</td>
<td>&nbsp;</td>
<td>GE Healthcare</td>
<td>BR-1004-11</td>
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the importance of correct protein concentration for kinetics and affinity determination in structure-function analysis 

في هذه الدراسة ، ونحن استكشاف التفاعل بين الابقار البروتيني السيستين باء cystatin المانع وشكل غير نشط من غراء حفاز (الشكل 1) ، وهو الأنزيم البروتيني السيستين النباتية ، في الوقت الحقيقي من خلال تحليل التسمية خالية باستخدام X100 Biacore. ويتم إنتاج العديد من المتغيرات B cystatin مع الطفرات نقطة في مجالات التفاعل مع غراء. لكل cystatin الخيار باء نحدد تركيزه ملزمة محددة باستخدام المعايرة خالية من التحليل تركيز (CFCA) وقارن بين القيم التي تم الحصول عليها مع تركيز البروتين الكلي وفقا لما يقرره<sub&gt; 280</sub&gt;. بعد ذلك ، يتم قياس حركية كل cystatin الخيار باء ملزم لاحد باستخدام غراء دورة حركية (SCK). نظهر أن واحدا من المتغيرات cystatin B الأربع الذي هو دراسة النشطة فقط جزئيا عن الربط. هذا النشاط الجزئي ، التي كشفت عنها CFCA ، يترجم إلى اختلاف كبير في معدل تكوين الجمعيات ثابت (<em&gt; ك</em<sub&gt; A</sub&gt;) وتقارب (<em&gt; K</em<sub&gt; D</sub&gt;) ، بالمقارنة مع مجموع القيم المحسوبة باستخدام تركيز البروتين. CFCA به بالاشتراك مع التحليل الحركي في دراسة بنية وظيفة تساهم في الحصول على نتائج موثوق بها ، ويساعد على جعل التفسير الصحيح لآلية التفاعل.

Watch this Scientific Journal Video about The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis at JoVE.com

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

نحن نطبق التسمية خالية من تحليل التفاعل البروتين باستخدام X100 Biacore للهيكل وظيفة تحليل الربط العديد من المسوخ B cystatin إلى غراء من خلال توصيف الحركية. المعايرة خالية من التحليل تركيز (CFCA) يقيس تركيز البروتين مع النشاط ملزم الاحتفاظ دون الحاجة إلى منحنى القياسية. نظهر أن تأكيد باستخدام تركيزات CFCA يزيد من مصداقية هذا التحليل الحركي ويمكن أن يعتمد عليه أن يحدد الثوابت الحركية حتى إذا تم خفض نشاط بروتين المؤتلف.

على أهمية تركيز البروتين الصحيح لحركية وتقارب في تحديد هيكل وظيفة التحليل

In this study, we explore the interaction between the bovine cysteine protease inhibitor cystatin B and a catalytically inactive form of papain (Fig. 1), ...

the-importance-correct-protein-concentration-for-kinetics-affinity

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Biology

The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis 

20.5K Views.  GE Healthcare Bio-Sciences AB. نحن نطبق التسمية خالية من تحليل التفاعل البروتين باستخدام X100 Biacore للهيكل وظيفة تحليل الربط العديد من المسوخ B cystatin إلى غراء من خلال توصيف الحركية. المعايرة خالية من التحليل تركيز (CFCA) يقيس تركيز البروتين مع النشاط ملزم الاحتفاظ دون الحاجة إلى منحنى القياسية. نظهر...

Video: The Importance of Correct Protein Concentration for Kinetics and Affinity Determination in Structure-function Analysis

Microvolume Protein Concentration Determination using the NanoDrop 2000c Spectrophotometer

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often used for protein analysis. The Thermo Scientific NanoDrop 2000c Spectrophotometer solves this issue with an innovative sample retention system that holds microvolume samples between two measurement surfaces using the surface tension properties of liquids, enabling the quantification of samples in volumes as low as 0.5-2 &mu;L. The elimination of cuvettes or capillaries allows real time changes in path length, which reduces the measurement time while greatly increasing the dynamic range of protein concentrations that can be measured. The need for dilutions is also eliminated, and preparations for sample quantification are relatively easy as the measurement surfaces can be simply wiped with laboratory wipe. This video article presents modifications to traditional protein concentration determination methods for quantification of microvolume amounts of protein using A280 absorbance readings or the BCA colorimetric assay.

Microvolume samples are quantified by a spectrophotometer system that uses natural surface tension to retain samples without the use of cuvettes or capillaries. The dynamic range of protein concentrations and speed by which they can be measured are greatly increased with this method.

Microvolume تحديد تركيز البروتين باستخدام 2000c NanoDrop الطيف

Traditional spectrophotometry requires placing samples into cuvettes or capillaries. This is often impractical due to the limited sample volumes often ...

Purification of the M. magneticum Strain AMB-1 Magnetosome Associated Protein MamA&Delta;41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is believed to aid their search for suitable environments (1). This capability is conferred by the magnetosome, a subcellular organelle that consists of a linear-chain assembly of lipid vesicles each able to biomineralize and enclose a ~50-nm crystal of magnetite or greigite. A principle component of the magnetosome that was shown to be required for the formation of functional vesicles is MamA. MamA is a highly abundant magnetosome-associated protein which is one of the most characterized magnetosome-associated proteins in vivo (2-6). This article focuses on the purification of MamA, which despite being studied in vivo, no clear functional or structural details have been identified for it. Bioinformatics analysis suggested that MamA is a tetra-tricopeptide repeat (TPR) containing protein. TPR is a structural motif found as such or forming part of a bigger fold in a wide range of proteins, it serves as a template for protein-protein interactions and mediates multi-protein complexes (7). TPRs are involved in many crucial tasks in eukaryotic cell organelle processes and many bacterial pathways (8-14). In order to understand MamA, a unique TPR containing protein, highly purified protein is required as a first step. In this article, we present the purification protocol for a stable MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

Purification of the &lt;em&gt;M. magneticum&lt;/em&gt; Strain AMB-1 Magnetosome Associated Protein MamA&amp;Delta;41

MamA is a unique Magnetosome associated protein which was shown to be involved in magnetosome activation. Here we present the purification protocol of MamA deletion mutant (MamA&Delta;41) from M. magneticum AMB-1.

تنقية م. magneticum سلالة AMB - 1 بروتين المرتبطين Magnetosome MamAΔ41

Magnetotactic bacteria comprise a diverse group of aquatic microorganisms that are able to orientate themselves along geomagnetic fields. This behavior is ...

Analysis of the Development of a Morphological Phenotype as a Function of Protein Concentration in Budding Yeast

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae. Although this protocol is based on the overexpression of a protein, it can easily be adapted for morphological phenotypes dependent on suppression of protein expression. Our lab is interested in studying the signaling properties of the endocytic adaptor protein epsin. To that purpose we used a dominant negative approach in which we over-expressed the conserved Epsin N-Terminal Homology (ENTH) domain in order to interfere with the functions of endogenous epsin-2 (Ent2 or YLR206W). We observed that overexpression of the ENTH domain of Ent2 (ENTH2) in wild type cells led to a cell division defect that is dependent on the mislocalization of a family of scaffolding proteins, septins.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of phenotype development as a function of protein concentration at population and single-cell levels in Saccharomyces cerevisiae.

تحليل للوضع النمط الظاهري المورفولوجية كدالة للتركيز البروتين في الخميرة الناشيء.

Gene deletion and protein overexpression are common methods for studying functions of proteins. In this article, we describe a protocol for analysis of ...

Analysis of mRNA Nuclear Export Kinetics in Mammalian Cells by Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although the nuclear export of mRNA has been studied extensively in Xenopus oocytes1 and genetically tractable organisms such as yeast2 and the Drosophila derived S2 cell line3, few studies had been conducted in mammalian cells. Furthermore the kinetics of mRNA export in mammalian somatic cells could only be inferred indirectly4,5. In order to measure the nuclear export kinetics of mRNA in mammalian tissue culture cells, we have developed an assay that employs the power of microinjection coupled with fluorescent in situ hybridization (FISH). These assays have been used to demonstrate that in mammalian cells, the majority of mRNAs are exported in a splicing dependent manner6,7, or in manner that requires specific RNA sequences such as the signal sequence coding region (SSCR) 6. In this assay, cells are microinjected with either in vitro synthesized mRNA or plasmid DNA containing the gene of interest. The microinjected cells are incubated for various time points then fixed and the sub-cellular localization of RNA is assessed using FISH. In contrast to transfection, where transcription occurs several hours after the addition of nucleic acids, microinjection of DNA or mRNA allows for rapid expression and allows for the generation of precise kinetic data.

Here we describe an assay that employs the power of microinjection coupled with fluorescent in situ hybridization in order to accurately measure the nuclear export kinetics of mRNA in mammalian somatic cells.

تحليل حركية مرنا الصادرات النووية في خلايا الثدييات التي Microinjection

In eukaryotes, messenger RNA (mRNA) is transcribed in the nucleus and must be exported into the cytoplasm to access the translation machinery. Although ...

Concentration Determination of Nucleic Acids and Proteins Using the Micro-volume Bio-spec Nano Spectrophotometer

Nucleic Acid quantitation procedures have advanced significantly in the last three decades. More and more, molecular biologists require consistent small-volume analysis of nucleic acid samples for their experiments. The BioSpec-nano provides a potential solution to the problems of inaccurate, non-reproducible results, inherent in current DNA quantitation methods, via specialized optics and a sensitive PDA detector. The BioSpec-nano also has automated functionality such that mounting, measurement, and cleaning are done by the instrument, thereby eliminating tedious, repetitive, and inconsistent placement of the fiber optic element and manual cleaning.
In this study, data is presented on the quantification of DNA and protein, as well as on measurement reproducibility and accuracy. Automated sample contact and rapid scanning allows measurement in three seconds, resulting in excellent throughput. Data analysis is carried out using the built-in features of the software. The formula used for calculating DNA concentration is:
Sample Concentration = DF &middot; (OD260-OD320)&middot; NACF (1)
Where DF = sample dilution factor and NACF = nucleic acid concentration factor.
The Nucleic Acid concentration factor is set in accordance with the analyte selected1.
Protein concentration results can be expressed as &mu;g/ mL or as moles/L by entering e280 and molecular weight values respectively. When residue values for Tyr, Trp and Cysteine (S-S bond) are entered in the e280Calc tab, the extinction coefficient values are calculated as e280 = 5500 x (Trp residues) + 1490 x (Tyr residues) + 125 x (cysteine S-S bond). The e280 value is used by the software for concentration calculation.
In addition to concentration determination of nucleic acids and protein, the BioSpec-nano can be used as an ultra micro-volume spectrophotometer for many other analytes or as a standard spectrophotometer using 5 mm pathlength cells.

This communication presents data on the accuracy and reproducibility of the BioSpec-nano UV-VIS spectrophotometer for dsDNA and protein quantitation. Even with ultra-small volumes (1 to 2 L), reproducibility is excellent, while the automated wiping function improves throughput and results in minimal carryover for more precise results.

تعيين تركيز الأحماض النووية والبروتينات باستخدام الصغرى ، حجم الطيف نانو بيو المواصفات

Nucleic Acid quantitation procedures have advanced significantly in the last three decades.  More and more, molecular biologists require consistent ...

A Liquid Phase Affinity Capture Assay Using Magnetic Beads to Study Protein-Protein Interaction: The Poliovirus-Nanobody Example

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein labeled, this method can be implemented for the investigation of protein-protein interactions. It is based on one hand on the recognition of the tagged protein by cobalt coated magnetic beads and on the other hand on the interaction between the tagged protein and a second specific protein that is labeled. First, the labeled and tagged proteins are mixed and incubated at room temperature. The magnetic beads, that recognize the tag, are added and the bound fraction of labeled protein is separated from the unbound fraction using magnets. The amount of labeled protein that is captured can be determined in an indirect way by measuring the signal of the labeled protein remained in the unbound fraction. The described liquid phase affinity assay is extremely useful when conformational conversion sensitive proteins are assayed. The development and application of the assay is demonstrated for the interaction between poliovirus and poliovirus recognizing nanobodies1. Since poliovirus is sensitive to conformational conversion2 when attached to a solid surface (unpublished results), the use of ELISA is limited and a liquid phase based system should therefore be preferred. An example of a liquid phase based system often used in polioresearch3,4 is the micro protein A-immunoprecipitation test5. Even though this test has proven its applicability, it requires an Fc-structure, which is absent in the nanobodies6,7. However, as another opportunity, these interesting and stable single-domain antibodies8 can be easily engineered with different tags. The widely used (His)6-tag shows affinity for bivalent ions such as nickel or cobalt, which can on their turn be easily coated on magnetic beads. We therefore developed this simple quantitative affinity capture assay based on cobalt coated magnetic beads. Poliovirus was labeled with 35S to enable unhindered interaction with the nanobodies and to make a quantitative detection feasible. The method is easy to perform and can be established with a low cost, which is further supported by the possibility of effectively regenerating the magnetic beads.

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. It is a reliable technique based on the interaction between magnetic beads and tagged proteins (e.g. nanobodies) on one hand and the affinity between the tagged protein and a second, labeled protein (e.g. poliovirus) on the other.

تقارب المرحلة السائلة الفحص التقاطها باستخدام الخرز المغناطيسي لدراسة البروتين البروتين التفاعل: المثال فيروس شلل الأطفال، Nanobody

In this article, a simple, quantitative, liquid phase affinity capture assay is presented. Provided that one protein can be tagged and another protein ...

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in current research and a plethora of methods for their investigation is available1. Protein-protein interactions often are highly dynamic and may depend on subcellular localization, post-translational modifications and the local protein environment2. Therefore, they should be investigated in their natural environment, for which co-immunoprecipitation approaches are the method of choice3. Co-precipitated interaction partners are identified either by immunoblotting in a targeted approach, or by mass spectrometry (LC-MS/MS) in an untargeted way. The latter strategy often is adversely affected by a large number of false positive discoveries, mainly derived from the high sensitivity of modern mass spectrometers that confidently detect traces of unspecifically precipitating proteins. A recent approach to overcome this problem is based on the idea that reduced amounts of specific interaction partners will co-precipitate with a given target protein whose cellular concentration is reduced by RNAi, while the amounts of unspecifically precipitating proteins should be unaffected. This approach, termed QUICK for QUantitative Immunoprecipitation Combined with Knockdown4, employs Stable Isotope Labeling of Amino acids in Cell culture (SILAC)5 and MS to quantify the amounts of proteins immunoprecipitated from wild-type and knock-down strains. Proteins found in a 1:1 ratio can be considered as contaminants, those enriched in precipitates from the wild type as specific interaction partners of the target protein. Although innovative, QUICK bears some limitations: first, SILAC is cost-intensive and limited to organisms that ideally are auxotrophic for arginine and/or lysine. Moreover, when heavy arginine is fed, arginine-to-proline interconversion results in additional mass shifts for each proline in a peptide and slightly dilutes heavy with light arginine, which makes quantification more tedious and less accurate5,6. Second, QUICK requires that antibodies are titrated such that they do not become saturated with target protein in extracts from knock-down mutants.
Here we introduce a modified QUICK protocol which overcomes the abovementioned limitations of QUICK by replacing SILAC for 15N metabolic labeling and by replacing RNAi-mediated knock-down for affinity modulation of protein-protein interactions. We demonstrate the applicability of this protocol using the unicellular green alga Chlamydomonas reinhardtii as model organism and the chloroplast HSP70B chaperone as target protein7 (Figure 1). HSP70s are known to interact with specific co-chaperones and substrates only in the ADP state8. We exploit this property as a means to verify the specific interaction of HSP70B with its nucleotide exchange factor CGE19.

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

We present a variation of the QUICK (QUantitative Immunoprecipitation Combined with Knockdown) approach that was introduced previously to distinguish between true and false protein-protein interactions. Our approach is based on 15N metabolic labeling, the modulation of affinities of protein-protein interactions by the presence/absence of ATP, immunoprecipitation, and quantitative mass spectrometry.

بروتوكول لتحديد البروتين البروتين التفاعلات على أساس<sup&gt; 15</sup&gt; N صفها الأيض، مناعي، الكمي والتحوير الطيف الكتلي الانجذاب

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

Protein Purification-free Method of Binding Affinity Determination by Microscale Thermophoresis

Quantitative characterization of protein interactions is essential in practically any field of life sciences, particularly drug discovery. Most of currently available methods of KD determination require access to purified protein of interest, generation of which can be time-consuming and expensive. We have developed a protocol that allows for determination of binding affinity by microscale thermophoresis (MST) without purification of the target protein from cell lysates. The method involves overexpression of the GFP-fused protein and cell lysis in non-denaturing conditions. Application of the method to STAT3-GFP transiently expressed in HEK293 cells allowed to determine for the first time the affinity of the well-studied transcription factor to oligonucleotides with different sequences. The protocol is straightforward and can have a variety of application for studying interactions of proteins with small molecules, peptides, DNA, RNA, and proteins.

Microscale thermophoresis (MST) can be widely used for determination of binding affinity without purification of the target protein from cell lysates. The protocol involves overexpression of the GFP-fused protein, cell lysis in non-denaturing conditions, and detection of MST signal in the presence of varying concentrations of the ligand.

البروتين طريقة خالية من تنقية تجليد تحديد الانجذاب من قبل Thermophoresis الميكروسكيل

Quantitative characterization of protein  interactions is essential in practically any field of life sciences,  particularly drug discovery. Most of ...

A Protocol for Phage Display and Affinity Selection Using Recombinant Protein Baits

Using recombinant phage as a scaffold to present various protein portions encoded by a directionally cloned cDNA library to immobilized bait molecules is an efficient means to discover interactions. The technique has largely been used to discover protein-protein interactions but the bait molecule to be challenged need not be restricted to proteins. The protocol presented here has been optimized to allow a modest number of baits to be screened in replicates to maximize the identification of independent clones presenting the same protein. This permits greater confidence that interacting proteins identified are legitimate interactors of the bait molecule. Monitoring the phage titer after each affinity selection round provides information on how the affinity selection is progressing as well as on the efficacy of negative controls. One means of titering the phage, and how and what to prepare in advance to allow this process to progress as efficiently as possible, is presented. Attributes of amplicons retrieved following isolation of independent plaque are highlighted that can be used to ascertain how well the affinity selection has progressed. Trouble shooting techniques to minimize false positives or to bypass persistently recovered phage are explained. Means of reducing viral contamination flare up are discussed.

Phage display is a powerful technique to capture proteins or protein moieties that interact with an immobilized molecule of interest. Once a decision of the type of phage cDNA library to create and screen has been made, the protocol described here permits efficient affinity selection leading to identification of interactors.

بروتوكول لفج العرض وتقارب اختيار طريق الطعوم البروتين المؤتلف

Using  recombinant phage as a scaffold to present various protein portions encoded by  a directionally cloned cDNA library to immobilized bait molecules ...

SpOT the Correct Tissue Every Time in Multi-tissue Blocks

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue microarrays make up the majority of multi-tissue paraffin blocks, but increasingly, researchers are using non-arrayed blocks containing larger tissues from multiple individuals which can provide many of the advantages of tissue microarrays without substantial investment in planning and equipment. A critical component of any multi-tissue analysis is the orientation method used to identify each individual tissue. Although methods exist to maintain proper orientation and identification of tissues in multi-tissue blocks, most are not well-suited to non-arrayed blocks, may consume valuable space within an array and/or are difficult to produce in the standard histology laboratory. The Specimen Orientation Tag (SpOT) is a simple, low cost orientation tool that is clearly visible in paraffin blocks and all tissue sections for reliable specimen identification in arrayed and non-arrayed layouts. The SpOT provides advantages over existing orientation methods for non-arrayed blocks as it does not require any direct modification to the tissue and allows for flexibility in the arrangement of tissue pieces.

The purpose of the Specimen Orientation Tag (SpOT) is to function as an orientation tool to aid in individual tissue identification in multi-tissue paraffin blocks. These protocols demonstrate how it is constructed easily from common, low-cost histology materials and serves as a reliable visual marker in paraffin blocks and sections.

بقعة الأنسجة الصحيحة في كل مرة في كتل متعددة النسيج

Multi-tissue paraffin blocks provide high throughput analysis with increased efficiency, experimental uniformity, and reduced time and cost. Tissue ...