Bu eser NIH R01 EB014869 tarafından desteklenmiştir.

1. hücre görüntüleme için hazırlanması
Not: Doğrudan gücü probun (DFP) herhangi bir yapisan hücre türü için kullanılabilir. Burada, NIH 3T3 fare fibroblastlar modeli hücre satır olarak bu iletişim kuralı için kullanılır.
<ol><li>Kültür NIH 3T3 fibroblast hücreleri içinde Dulbecco'nın modifiye kartal Orta (% 10 donör Sığır serum ve % 1 ile DMEM) penisilin-streptomisin 35-mm cam alt çanağı kadar istenen confluency. 37 ° C ve % 5 CO2hücreleri korumak.<ol>
<li>Tüm 35-mm cam alt yemekler 5 µg/mL fibronektin (veya benzer ECM protein) ile NIH 3T3 hücreleri görüntüleme için tohum önce kat emin olun. Not: Tam hücreleri yaymak gerekir ve yemek için deneme üzerinde yapışık. DFP yöntemin işe yaraması confluency açısından herhangi bir kısıtlama yoktur.</li>
	</ol>
</li>
	<li>Deney hemen önce hücreleri iki kez tam büyüme orta ile tek bir yıkama ardından PBS ile yıkayın.</li>
	<li>Tam büyüme orta 3 mL Cam alt yemek için ekleyin.</li>
</ol>2. mikroskobu ve resim alma
Not: Bir ters floresans mikroskobu (veya eşdeğeri) ile üreticinin önerilerini göre yan kol için yüklü micromanipulator. Mikroskop da 37 ° C sıcaklıkta ve % 5 CO2 düzeyinde korumak için çevre bir odası ile outfitted. Bir micromanipulator ve microinjector için mikroskop bağlı gereklidir de. Bir petrol daldırma 40 x / 1.3 NA veya 60 x / 1,49 NA (ya da eşdeğer hedefleri) deneyler için tavsiye edilir. Mikroskop bir titreşim yalıtım masaya monte edilmelidir.
<img alt="Figure 1" class="xfigimg" src="/files/ftp_upload/58038/58038fig1.jpg"> Resim 1 . Nükleer deformasyon ve mikroskop odaklanarak A. en büyük nükleer deformasyon ve nükleer deformasyon gevşeme. En büyük nükleer deformasyon hesaplamadan önce nükleer şekillerin arka kenarları ilk deforme çekirdeği çeviri için düzeltmek için çakıştı. Micropipette ipucu dekolmanı Şu anda çekirdeğin şekli ilk nükleer şeklin üzerine çekerek önce overlaid. Alan iki şekil arasındaki farkı Δ ölçülmüştürbir1. En büyük nükleer deformasyon ΔA1 orijinal nükleer alanına göre bölünmüş olarak tanımlanmıştır. Benzer şekilde, ikinci bir parametre, ΔA2, son kararlı duruma nükleer şekli özgün nükleer şeklindeki micropipette dekolmanı sonra overlaying tarafından tanımlı. B. hücre uçak A'da odaklanmak ve odak düzlemi micropipette ipucu bulmak için uçak B kadar hareket ettirin. Görüntüleme sırasında micropipette sağa (turuncu ok yönünü) çevrildi. Bu rakam Neelam ve arkdeğiştirildi. 15. <a href="https://www.jove.com/files/ftp_upload/58038/58038fig1large.jpg" target="_blank">Bu rakam daha büyük bir versiyonunu görüntülemek için buraya tıklayınız.</a>
<ol><li>Üreticinin iletişim kuralı başına microinjector açın.</li>
	<li>Bir daldırma yağ damlalık kullanmak, Daldırma yağ objektif lens üzerinde tek bir damla uygulanır.</li>
	<li>Çanak sıkıca çanak tutucusuna klamp ve çanak tutucu sahne alanı'na yük. Not: Hücreleri 37 ° C ve % 5 CO2 deneme boyunca muhafaza edilmelidir.</li>
	<li>Hücreleri (Uçak A, Şekil 1B) odak haline getirmek için hedefi yüksekliğini ayarlayın.</li>
	<li>Mikroskop sahne alanı ilgi bir hücreyi bulmak için hareket ederler.</li>
	<li>Oyun çubuğu damlalıklı tutucu üst konuma taşımak için micromanipulator üzerinde döndürün. 0.5 µm çapı uç micropipette damlalıklı tutucu üzerine yükleyin.<ol>
<li>Micropipette için hücre adezyon önlemek için oda sıcaklığında 0.3 mg/mL PLL-g-PEG çözüm 1 h için micropipette ucuyla önceden tedavi. Yapışma için emme herhangi bir baskı olmadan çekirdeği için micropipette dokunmadan da çekirdek uzak micropipette çeviri sınayın. Yapışma yokluğu nükleer deformasyon ve çeviri tam bir eksikliği ayırt edilebilir. Not: Lütfen paketi açmak için üretim önerileri uygulayın.</li>
	</ol>
</li>
	<li>Objektif odak düzlemi uçak A ve uçak b hücrenin üstüne üzerinde daha iyi denetim ayarlayarak yükseltmek (Şekil 1B, bkz: adım 2.4).</li>
	<li>Micromanipulator kaba denetimine ayarlayın. Tamamen odak haline gelene kadar micropipette micropipette, siluet için izleyerek micropipette uçak B aşağı yavaşça.</li>
	<li>Micropipette ipucu odak olduğunda, micromanipulator iyi denetimine ayarlayın.</li>
	<li>(Uçak A, Şekil 1B) hücrenin Ekvator uçak amaca daha düşük ve micropipette yaklaşık 15 µm (Şekil 1B, kesik micropipette) Uçak A yukarıda için daha düşük.</li>
	<li>Tazminat basınç (Pc) istenilen basınç için microinjector olarak ayarlayın; Basıncı dengelemek için birkaç saniye bekleyin. Not: Optimum basınç ayar noktası hücre türüne ve denemenin belirli hedeflere göre değişir. Çoğu durumda, 300 hPa iyi bir başlangıç noktası olur.</li>
	<li>Micropipette micromanipulator panelde temiz ayarı kullanarak ve hava kabarcıkları micropipette ucundan ortaya emin olmak için kontrol tıkalı değil emin olun.</li>
	<li>Ucu ucunu hafifçe nükleer yüzey dokunuyor kadar yavaş yavaş micropipette düşürerek hücreye yerleştirin. Not: ne zaman micropipette düşürücü, micropipette ucunu silüeti odak haline gelir olarak açık olacak. Micropipette çekirdek dokunamaz önce objektif odak yükseltmek ve micropipette çekirdeği (aynı x-y koordinatı, daha yüksek z-uçak) ile hizalayın. Odak (uçak A, Şekil 1B) çekirdeği Ekvator uçağa geri dönmek ve yavaş yavaş micropipette ucunu alt.</li>
	<li>Bir mühür micropipette uç ve nükleer membran arasında böylece atmosfer micropipette Tüp sonuna açılış mikroenjeksiyon sisteminden basınç kaynağı tüp keserek oluşturun. Bu adım bir negatif basınç nükleer yüzeyde eşit Pc oluşturur.</li>
	<li>Mikroskoplar resim koleksiyonu yazılımı ile görüntüleri elde etmek. AVI-edinme (video) veya nd-edinme (görüntüleri) görüntü collection yazılım ayarlayın. Not: yazılım edinme herhangi bir görüntüleme için gerçek zamanlı video görüntüleme veya Hızlandırılmış görüntü satın almalar ile bir kısa süre aralığı ayarlayın.</li>
	<li>Kanal görüntüleme karşılık gelen floresan için geçiş (Yani, GFP, RFP, vb) ve görüntüleme başlar.</li>
	<li>Micropipette İpucu (sağa doğru Şekil 1B) hücre gövdesinden çekin çevirmek kadar çekirdek micropipette ayırır. Not: ucu pozitif x-yön boyunca (görüş alanı sağa) çekin. Programlanmış ve bilgisayar tarafından kontrol çekme oranı veya joystick el ile hareket edebilir. Biz çekerek arasında herhangi bir korelasyon bulunmadı oranı ve nükleer deformasyon15 zorlamak için öncelikle elastik bir yanıt düşündüren.</li>
</ol>3. veri analizi
<ol><li>Herhangi bir temel görüntü işleme yazılımı ile görüntü analizi gerçekleştirin. Nükleer deformasyon ölçüde uzunluğu zorlanma (Ɛ) ya da (Şekil 1A) alan zorlanma sayılabilir. Denklem 1, kullanarak uzunluğu zorlanma ölçmek nerede L ve M0 temsil eden en fazla deformasyon ve ilk konum, çekirdeği uzunlukları sırasıyla. <img alt="Equation 1" src="/files/ftp_upload/58038/58038eq1.jpg">(Denklem 1)</li>
</ol>

<ol>
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Yazarlar ifşa gerek yok.

(Çekirdek nerede sitoiskeleti bilgisini iletmiyor) her iki izole çekirdeği gerektirdiğinden sitoiskeleti çekirdekle mekanik entegrasyonu ölçme micropipette aspirasyon16gibi en yeni yöntemleri için bir meydan okuma olduğunu veya çekirdekleri askıya alınmış hücrelerindeki (hücre dışı güçleri, çekiş güçleri gibi eksik durumda). Güç çekirdeğine hücre yapışık bir membran17,18biaxial baskı uygulayarak uygulanm?...

Patolojiler kanser gibi nükleer şekli ve yapısı1,2, genellikle bir 'yumuşama' çekirdek3,4tarafından yayımlanır değişiklikleri içerir. Nükleer mekanik deformasyon direnci genellikle izole çekirdeği5' e bir kuvvet uygulayarak karakterize.
Hücre çekirdeğinde tatlı sitoiskeleti için bağlayıcı Nucleoskeleton ve sitoiskeleti (LINC) karmaşık6,7,8,9tarafından bağlanır. Sonuç olarak, çekirdek mekanik sitoiskeleti ile ve hücre-terkedilemeyen yapışıklıklar, hücre dışı matriks ile entegre edilmiştir. Mekanik yapışık hücreleri içinde çekirdek sondalama mekanik Bu entegrasyon içgörü sağlayabilir. Micropipette aspirasyon10,11ve atomik kuvvet mikroskobu12,13,14çekirdeği canlı hücreler içinde işlemek için yöntemler içerir. Biz son zamanlarda bir yaşam yapisan hücre15çekirdeğinde doğrudan mekanik Kuvvetleri uygulandığı bir doğrudan kuvvet sonda (DFP) nitelendirdi.
Burada, mikroskobu tesislerinde doğrudan yapisan bir hücre çekirdeğinde için bilinen, nano ölçekli mekanik kuvvet uygulamak için yaygın olarak kullanılabilir bir mikroenjeksiyon sistemi kullanan yordamı anahat. Bir femtotip (0.5 µm çapı micropipette tıp) monte ve bir tüp tarafından mikroenjeksiyon sisteme bağlı. Kültür çanak yüzeyine göre 45 ° açıyla konumlandırılmış uç, bitişik kadar nükleer yüzeye indirilir. Tüp sonra bağlantısı kesilen ve nükleer yüzeyinde negatif bir emme basınç oluşturur ve nükleer yüzeye karşı micropipette ipucu mühürler atmosfere açtı. Çeviri micropipette uç çekirdeği deforme ve sonunda (büyüklüğü yük bağlı olarak), micropipette ilişkisi kesildi. Bu dekolmanı çekirdek ve hücre, tarafından sarf geri yükleme (dirençli) kuvvetleri micropipette tarafından uygulanan emiş gücü eşit oluşur. Çözümleme yapılır çekirdeği, deplasman ölçerek uzunluğu zorlanma (denklem 1) veya alan zorlanma (Şekil 1A).

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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">FluoroDish</td>
			<td style="width:71px;">WPI</td>
			<td style="width:119px;">FD35</td>
			<td style="width:355px;"></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">SYTO 59</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td style="width:119px;">S11341</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Femtotips&#160;</td>
			<td style="width:71px;">Eppendorf</td>
			<td align="right">930000043</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">InjectMan NI2</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>discontinued, current equivalent model: InjectMan 4</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">FemtoJet</td>
			<td style="width:71px;">Eppendorf</td>
			<td style="width:119px;">NA</td>
			<td>Current model FemtoJet 4i</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Plan Fluor oil immersion 40x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Apo TIRF oil immersion 60x</td>
			<td style="width:71px;">Nikon</td>
			<td style="width:119px;">NA</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:299px;">Donor Bovine Serum (DBS)</td>
			<td style="width:71px;">ThermoFisher Scientific</td>
			<td align="right" style="width:119px;">16030074</td>
			<td>NIH 3T3 serum</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Dulbecco&#39;s Modification of Eagle&#39;s (DMEM)</td>
			<td style="width:71px;">Mediatech cellgro</td>
			<td style="width:119px;">MT10013CVRF</td>
			<td>NIH 3T3 medium</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:299px;">Penicillin-Streptomycin&#160;</td>
			<td style="width:71px;">Mediatech</td>
			<td style="width:119px;">MT30004CIRF</td>
			<td>NIH 3T3 medium supplement</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:299px;">Immersion Oil Type LDF Non-Fluorescing</td>
			<td style="width:71px;">Nikon</td>
			<td align="right" style="width:119px;">77007</td>
			<td>Immersion oil for objective lens&#160;</td>
		</tr>
	</tbody>
</table>

a direct force probe for measuring mechanical integration between the nucleus and the cytoskeleton

Çekirdeği mekanik özellikleri mekanik Kuvvetleri hücrelerinde üretilen onun yanıt belirler. Çekirdek ile sitoiskeleti tatlı sürekli olduğundan, yöntemleri mekanik davranışını yapisan hücrelerdeki soruşturma için ihtiyaç vardır. Burada, doğrudan gücü probun (DFP) doğrudan bir yaşam yapisan Hücre çekirdeğinde kuvvet uygulamak için bir araç olarak tartışmak. Emme nükleer yüzeye dar bir micropipette ekleyin. Micropipette deforme ve çevirmek çekirdek neden olan çekirdek uzak çevrilir. Geri yükleme kuvvetleri emiş gücüne eşit olduğunda, çekirdek ayırır ve Melosun rahatlatır. Emiş basıncı kesin olarak bilindiğinden, güç nükleer yüzeyi bilinir. Bu yöntem nano ölçekli güçleri deforme ve yapışık Hücre çekirdeğinde çevirmek için yeterli ve kuvvetler direnmeye çekirdek etkinleştirmek hücre iskeleti elemanlar ortaya koymuştur. DFP hücresel ve nükleer bileşenleri nükleer mekanik özellikleri canlı hücreler'katkılarıyla incelemek için kullanılabilir.

Şekil 2A bir NIH 3T3 fare fibroblast çekirdeği zorlayarak gösterir. Micropipette ucunu sağa çevrilir, çekirdeği deforms ve sonunda micropipette ucundan ayırır. Çekirdek uzunluğu suşu emiş gücü (Şekil 2B) ile artacaktır görülmektedir. Çekirdeği (kenar çekerek micropipette) ön kenarındaki nükleer bir çıkıntı oluşturan ve trailing edge özgün konumundan yerinden olduğunu. Çıkıntı uzunluğu çekir...

Watch this Scientific Journal Video about A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton at JoVE.com

A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Çekirdek ve sitoiskeleti mekanik bütünleşmesi ölçmek için bir doğrudan kuvvet sonda

Bu protokol için doğrudan bir yaşam Hücre çekirdeğinde kontrollü bir kuvvet uygulamak için micropipette yöntemi açıklanmaktadır. Bu tahlil sorgulama yaşayan, yapisan hücredeki nükleer mekanik özellikleri sağlar.

The mechanical properties of the nucleus determine its response to mechanical forces generated in cells. Because the nucleus is molecularly continuous ...

a-direct-force-probe-for-measuring-mechanical-integration-between

Research

JoVE Journal

Biochemistry

15.1K Görüntüleme.  University of Florida. Bu protokol için doğrudan bir yaşam Hücre çekirdeğinde kontrollü bir kuvvet uygulamak için micropipette yöntemi açıklanmaktadır. Bu tahlil sorgulama yaşayan, yapisan hücredeki nükleer mekanik özellikleri sağlar.

Video: A Direct Force Probe for Measuring Mechanical Integration Between the Nucleus and the Cytoskeleton

Method for Identifying Small Molecule Inhibitors of the Protein-protein Interaction Between HCN1 and TRIP8b

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the excitability of neurons. The subcellular distribution of these channels in pyramidal neurons of hippocampal area CA1 is regulated by tetratricopeptide repeat-containing Rab8b interacting protein (TRIP8b), an auxiliary subunit. Genetic knockout of HCN pore forming subunits or TRIP8b, both lead to an increase in antidepressant-like behavior, suggesting that limiting the function of HCN channels may be useful as a treatment for Major Depressive Disorder (MDD). Despite significant therapeutic interest, HCN channels are also expressed in the heart, where they regulate rhythmicity. To circumvent off-target issues associated with blocking cardiac HCN channels, our lab has recently proposed targeting the protein-protein interaction between HCN and TRIP8b in order to specifically disrupt HCN channel function in the brain. TRIP8b binds to HCN pore forming subunits at two distinct interaction sites, although here the focus is on the interaction between the tetratricopeptide repeat (TPR) domains of TRIP8b and the C terminal tail of HCN1. In this protocol, an expanded description of a method for purifying TRIP8b and executing a high throughput screen to identify small molecule inhibitors of the interaction between HCN and TRIP8b, is described. The method for high throughput screening utilizes a Fluorescence Polarization (FP) -based assay to monitor the binding of a large TRIP8b fragment to a fluorophore-tagged eleven amino acid peptide corresponding to the HCN1 C terminal tail. This method allows &#39;hit&#39; compounds to be identified based on the change in the polarization of emitted light. Validation assays are then performed to ensure that &#39;hit&#39; compounds are not artifactual.

The interaction between HCN channels and their auxiliary subunit has been identified as a therapeutic target in Major Depressive Disorder. Here, a fluorescence polarization-based method for identifying small molecule inhibitors of this protein-protein interaction, is presented.

Yöntem HCN1 ve TRIP8b arasında protein-protein etkileşimi küçük molekül önleyicilerinin tespit edilmesi için

Hyperpolarization-activated cyclic nucleotide-gated (HCN) channels are expressed ubiquitously throughout the brain, where they function to regulate the ...

Biyokimya

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

Zaman çözülmesi elektro sprey iyonizasyon Hidrojen-döteryum Değişim Kütle Spektrometre Protein Yapısı ve Dinamikleri incelenmesi için

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

Force Spectroscopy of Single Protein Molecules Using an Atomic Force Microscope

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. Atomic force microscopy (AFM) can address this problem by enabling stretching and relaxation of single protein molecules, which gives direct evidence of specific unfolding and refolding characteristics. AFM-based single-molecule force-spectroscopy (AFM-SMFS) provides a means to consistently measure high-energy conformations in proteins that are not possible in traditional bulk (biochemical) measurements. Although numerous papers were published to show principles of AFM-SMFS, it is not easy to conduct SMFS experiments due to a lack of an exhaustively complete protocol. In this study, we briefly illustrate the principles of AFM and extensively detail the protocols, procedures, and data analysis as a guideline to achieve good results from SMFS experiments. We demonstrate representative SMFS results of single protein mechanical unfolding measurements and we provide troubleshooting strategies for some commonly encountered problems.

We describe the detailed procedures and strategies to measure the mechanical properties and mechanical unfolding pathways of single protein molecules using an atomic force microscope. We also show representative results as a reference for selection and justification of good single protein molecule recordings.

Kuvvet spektroskopisi bir Atomik kuvvet mikroskobu kullanılarak tek Protein moleküllerinin

The determination of the folding process of proteins from their amino acid sequence to their native 3D structure is an important problem in biology. ...

Utilizing a Comprehensive Immunoprecipitation Enrichment System to Identify an Endogenous Post-translational Modification Profile for Target Proteins

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which may be essential for a given protein&#39;s role both physiologically and pathologically. Enrichment of PTMs is often necessary when investigating the PTM status of a target protein, because PTMs are often transient and relatively low in abundance. Many pitfalls are encountered when enriching for a PTM of a target protein, such as buffer incompatibility, the target protein antibody is not IP-compatible, loss of PTM signal, and others. The degree of difficulty is magnified when investigating multiple PTMs like acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation for a given target protein. Studying a combination of these PTMs may be necessary, as crosstalk between PTMs is prevalent and critical for protein regulation. Often, these PTMs are studied in different lysis buffers and with unique inhibitor compositions. To simplify the process, a unique denaturing lysis system was developed that effectively isolates and preserves these four PTMs; thus, enabling investigation of potential crosstalk in a single lysis system. A unique filter system was engineered to remove contaminating genomic DNA from the lysate, which is a problematic by-product of denaturing buffers. Robust affinity matrices targeting each of the four PTMs were developed in concert with the buffer system to maximize the enrichment and detection of the endogenous states of these four PTMs. This comprehensive PTM detection toolset streamlines the process of obtaining critical information about whether a protein is modified by one or more of these PTMs.

Investigating multiple, endogenous post-translational modifications for a target protein can be extremely challenging. Techniques described here utilize an optimized lysis buffer and filter system developed with specific PTM-targeting, affinity matrices to detect the acetylation, ubiquitination, SUMOylation 2/3, and tyrosine phosphorylation modifications for a target protein using a single, streamlined system.

Bir endojen translasyonel modifikasyon profil hedef proteinler için tanımlamak için bir kapsamlı Immunoprecipitation zenginleştirme sistemi kullanan

It is now well-appreciated that post-translational modifications (PTMs) play an integral role in regulating a protein&#39;s structure and function, which ...

A Colorimetric Method for Measuring Iron Content in Plants

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content is rather low in all organisms, amounting in plants to about 0.009% of dry weight. To date, one of the most accurate methods for measuring iron concentration in plant tissues is flame absorption atomic spectroscopy. However, this approach is time-consuming and expensive and requires specific equipment not commonly found in plant laboratories. Therefore, a simpler, yet accurate method that can be routinely used is needed. The colorimetric Prussian Blue method is regularly used for qualitative iron staining in animal and plant histological sections. In this study, we adapted the Prussian Blue method for quantitative measurements of iron in tobacco leaves. We validated the accuracy of this method using both atomic spectroscopy and Prussian Blue staining to measure iron content in the same samples and found a linear regression (R2 = 0.988) between the two procedures. We conclude that the Prussian Blue method for quantitative iron measurement in plant tissues is precise, simple, and inexpensive. However, the linear regression presented here may not be appropriate for other plant species, due to potential interactions between the sample and the reagent. Establishment of a regression curve is thus needed for different plant species.

We present a simple and reliable protocol for measuring iron content in plant tissues using the colorimetric Prussian Blue method.

Demir içeriği tesislerinde ölçme kolorimetrik yöntemi

Iron, one of the most important micronutrients in living organisms, is involved in basic processes, such as respiration and photosynthesis. Iron content ...

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For a long time, the gene expression studies were dominated by research on the transcriptional level. However, the steady-state levels of mRNAs do not correlate well with protein production, and the translatability of mRNAs varies greatly depending on the conditions. In some organisms, like the parasite Leishmania, the protein expression is regulated mostly at the translational level. Recent studies demonstrated that protein translation dysregulation is associated with cancer, metabolic, neurodegenerative and other human diseases. Polysome profiling is a powerful method to study protein translation regulation. It allows to measure the translational status of individual mRNAs or examine translation on a genome-wide scale. The basis of this technique is the separation of polysomes, ribosomes, their subunits and free mRNAs during centrifugation of a cytoplasmic lysate through a sucrose gradient. Here, we present a universal polysome profiling protocol used on three different models - parasite Leishmania major, cultured human cells and animal tissues. Leishmania cells freely grow in suspension and cultured human cells grow in adherent monolayer, while mouse testis represents an animal tissue sample. Thus, the technique is adapted to all of these sources. The protocol for the analysis of polysomal fractions includes detection of individual mRNA levels by RT-qPCR, proteins by Western blot and analysis of ribosomal RNAs by electrophoresis. The method can be further extended by examination of mRNAs association with the ribosome on a transcriptome level by deep RNA-seq and analysis of ribosome-associated proteins by mass spectroscopy of the fractions. The method can be easily adjusted to other biological models.

Polysome Profiling in Leishmania, Human Cells and Mouse Testis

The overall goal of polysome profiling technique is analysis of translational activity of individual mRNAs or transcriptome mRNAs during protein synthesis. The method is important for studies of protein synthesis regulation, translation activation and repression in health and multiple human diseases.

Polysome LEISHMANIA, insan hücreleri ve fare Testis profil oluşturma

Proper protein expression at the right time and in the right amounts is the basis of normal cell function and survival in a fast-changing environment. For ...

Covalent Immobilization of Proteins for the Single Molecule Force Spectroscopy

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and functions. It gave us the opportunity to explore a multiplicity of biophysical mechanisms, e.g., how bacterial adhesins bind to host surface receptors in more detail. Among other factors, the success of SMFS experiments depends on the functional and native immobilization of the biomolecules of interest on solid surfaces and AFM tips. Here, we describe a straightforward protocol for the covalent coupling of proteins to silicon surfaces using silane-PEG-carboxyls and the well-established N-hydroxysuccinimid/1-ethyl-3-(3-dimethyl-aminopropyl)carbodiimid (EDC/NHS) chemistry in order to explore the interaction of pilus-1 adhesin RrgA from the Gram-positive bacterium Streptococcus pneumoniae (S. pneumoniae) with the extracellular matrix protein fibronectin (Fn). Our results show that the surface functionalization leads to a homogenous distribution of Fn on the glass surface and to an appropriate concentration of RrgA on the AFM cantilever tip, apparent by the target value of up to 20% of interaction events during SMFS measurements and revealed that RrgA binds to Fn with a mean force of 52 pN. The protocol can be adjusted to couple via site specific free thiol groups. This results in a predefined protein or molecule orientation and is suitable for other biophysical applications besides the SMFS.

This protocol describes the covalent immobilization of proteins with a heterobifunctional silane coupling agent to silicon-oxide surfaces designed for the atomic force microscopy based single molecule force spectroscopy which is exemplified by the interaction of RrgA (pilus-1 tip adhesin of S. pneumoniae) with fibronectin.

Proteinler kovalent immobilizasyon için tek molekül kuvvet spektroskopisi

In recent years, atomic force microscopy (AFM) based single molecule force spectroscopy (SMFS) extended our understanding of molecular properties and ...

Imaging of Extracellular Vesicles by Atomic Force Microscopy

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane proteins and other molecules, and diverse luminal content inherited from a parent cell, which includes RNAs, proteins, and DNAs. The characterization of the hydrodynamic sizes of EVs, which depends on the size of the vesicle and its coronal layer formed by surface decorations, has become routine. For exosomes, the smallest of EVs, the relative difference between the hydrodynamic and vesicles sizes is significant. The characterization of vesicles sizes by the cryogenic transmission electron microscopy (cryo-TEM) imaging, a gold standard technique, remains a challenge due to the cost of the instrument, the expertise required to perform the sample preparation, imaging and data analysis, and a small number of particles often observed in images. A widely available and accessible alternative is the atomic force microscopy (AFM), which can produce versatile data on three-dimensional geometry, size, and other biophysical properties of extracellular vesicles. The developed protocol guides the users in utilizing this analytical tool and outlines the workflow for the analysis of EVs by the AFM, which includes the sample preparation for imaging EVs in hydrated or desiccated form, the electrostatic immobilization of vesicles on a substrate, data acquisition, its analysis, and interpretation. The representative results demonstrate that the fixation of EVs on the modified mica surface is predictable, customizable, and allows the user to obtain sizing results for a large number of vesicles. The vesicle sizing based on the AFM data was found to be consistent with the cryo-TEM imaging.&#160;

A step-by-step procedure is described for label-free immobilization of exosomes and extracellular vesicles from liquid samples and their imaging by atomic force microscopy (AFM). The AFM images are used to estimate the size of the vesicles in the solution and characterize other biophysical properties.&#160;

Atomik Kuvvet Mikroskobu ile Hücre Dışı Veziküllerin Görüntülenmesi

Exosomes and other extracellular vesicles (EVs) are molecular complexes consisting of a lipid membrane vesicle, its surface decoration by membrane ...

High-Throughput Total Internal Reflection Fluorescence and Direct Stochastic Optical Reconstruction Microscopy Using a Photonic Chip

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced contrast due to optical sectioning. The conventional approach is to use high numerical aperture microscope TIRF objectives for both excitation and collection, severely limiting the field of view and throughput. We present a novel approach to generating TIRF excitation for imaging with optical waveguides, called chip-based nanoscopy. The aim of this protocol is to demonstrate how chip-based imaging is performed in an already built setup. The main advantage of chip-based nanoscopy is that the excitation and collection pathways are decoupled. Imaging can then be done with a low magnification lens, resulting in large field of view TIRF images, at the price of a small reduction in resolution. Liver sinusoidal endothelial cells (LSECs) were imaged using direct stochastic optical reconstruction microscopy (dSTORM), showing a resolution comparable to traditional super-resolution microscopes. In addition, we demonstrate the high-throughput capabilities by imaging a 500 &#181;m x 500 &#181;m region with a low magnification lens, providing a resolution of 76 nm. Through its compact character, chip-based imaging can be retrofitted into most common microscopes and can be combined with other on-chip optical techniques, such as on-chip sensing, spectroscopy, optical trapping, etc. The technique is thus ideally suited for high throughput 2D super-resolution imaging, but also offers great opportunities for multi-modal analysis.

Chip-based super-resolution optical microscopy is a novel approach to fluorescence microscopy and offers advantages in cost effectiveness and throughput. Here, the protocols for chip preparation and imaging are shown for TIRF microscopy and localization-based super-resolution microscopy.

Fotonik Çip Kullanarak Yüksek İşLeç Toplam İç Yansıma Floresansı ve Direkt Stokastik Optik Rekonstrüksiyon Mikroskobu

Total internal reflection fluorescence (TIRF) is commonly used in single molecule localization based super-resolution microscopy as it gives enhanced ...

A High-Throughput Enzyme-Coupled Activity Assay to Probe Small Molecule Interaction with the dNTPase SAMHD1

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this enzyme can hydrolyze dNTPs into their corresponding nucleosides and inorganic triphosphates. Due to its critical role in nucleotide metabolism, its association to several pathologies, and its role in therapy resistance, intense research is currently being carried out for a better understanding of both the regulation and cellular function of this enzyme. For this reason, development of simple and inexpensive high-throughput amenable methods to probe small molecule interaction with SAMHD1, such as allosteric regulators, substrates, or inhibitors, is vital. To this purpose, the enzyme-coupled malachite green assay is a simple and robust colorimetric assay that can be deployed in a 384-microwell plate format allowing the indirect measurement of SAMHD1 activity. As SAMHD1 releases the triphosphate group from nucleotide substrates, we can couple a pyrophosphatase activity to this reaction, thereby producing inorganic phosphate, which can be quantified by the malachite green reagent through the formation of a phosphomolybdate malachite green complex. Here, we show the application of this methodology to characterize known inhibitors of SAMHD1 and to decipher the mechanisms involved in SAMHD1 catalysis of non-canonical substrates and regulation by allosteric activators, exemplified by nucleoside-based anticancer drugs. Thus, the enzyme-coupled malachite green assay is a powerful tool to study SAMHD1, and furthermore, could also be utilized in the study of several enzymes which release phosphate species.

SAMHD1 is a deoxynucleoside triphosphate triphosphohydrolase with critical roles in human health and disease. Here we present a versatile enzyme-coupled SAMHD1 activity assay, deployed in a 384-well microplate format, that allows for the evaluation of small molecules and nucleotide analogues as SAMHD1 substrates, activators, and inhibitors.

dNTPase SAMHD1 ile Küçük Molekül Etkileşimini Araştırmak için Yüksek Verimli Enzime Bağlı Aktivite Testi

Sterile alpha motif and HD domain-containing protein 1 (SAMHD1) is a pivotal regulator of intracellular deoxynucleoside triphosphate (dNTP) pools, as this ...