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<ol>
	<li>Fazal FM, et al. Atlas of subcellular RNA localization revealed by APEX-seq. Cell. 2019;178(2):473-90.e26.</li>
	<li>Padron A, Iwasaki S, Ingolia NT. Proximity RNA labeling by APEX-seq reveals the organization of translation initiation complexes and repressive RNA granules. Mol Cell. 2019;75(4):875-87.e5.</li>
	<li>Dorner K, Ruggeri C, Zemp I, Kutay U. Ribosome biogenesis factors: from names to functions. EMBO J. 2023;42(7):e112699.</li>
	<li>Klinge S, Woolford JL Jr. Ribosome assembly coming into focus. Nat Rev Mol Cell Biol. 2019;20(2):116-31.</li>
	<li>Aviram N, Schuldiner M. Targeting and translocation of proteins to the endoplasmic reticulum at a glance. J Cell Sci. 2017;130(24):4079-85.</li>
	<li>Dahan N, et al. Peroxisome function relies on organelle-associated mRNA translation. Sci Adv. 2022;8(2):eabk2141.</li>
	<li>Koppers M, et al. Receptor-specific interactome as a hub for rapid cue-induced selective translation in axons. Elife. 2019;8:e48718.</li>
	<li>Hafner AS, et al. Local protein synthesis is a ubiquitous feature of neuronal pre- and postsynaptic compartments. Science. 2019;364(6441):eaau3644.</li>
	<li>Biever A, et al. Monosomes actively translate synaptic mRNAs in neuronal processes. Science. 2020;367(6477):eaay4991.</li>
	<li>Williams CC, Jan CH, Weissman JS. Targeting and plasticity of mitochondrial proteins revealed by proximity-specific ribosome profiling. Science. 2014;346(6210):748-51.</li>
	<li>Lesnik C, et al. OM14 is a mitochondrial receptor for cytosolic ribosomes that supports co-translational import into mitochondria. Nat Commun. 2014;5:5711.</li>
	<li>Gold VA, Chroscicki P, Bragoszewski P, Chacinska A. Visualization of cytosolic ribosomes on the surface of mitochondria by electron cryo-tomography. EMBO Rep. 2017;18(10):1786-800.</li>
	<li>Simsek D, et al. The mammalian ribo-interactome reveals ribosome functional diversity and heterogeneity. Cell. 2017;169(6):1051-65.e18.</li>
	<li>Kondrashov N, et al. Ribosome-mediated specificity in Hox mRNA translation and vertebrate tissue patterning. Cell. 2011;145(3):383-97.</li>
	<li>Shi Z, et al. Heterogeneous ribosomes preferentially translate distinct subpools of mRNAs genome-wide. Mol Cell. 2017;67(1):71-83.e7.</li>
	<li>Genuth NR, et al. A stem cell roadmap of ribosome heterogeneity reveals a function for RPL10A in mesoderm production. Nat Commun. 2022;13(1):5491.</li>
	<li>Li H, et al. A male germ-cell-specific ribosome controls male fertility. Nature. 2022;612(7941):725-31.</li>
	<li>Milenkovic I, et al. Dynamic interplay between RPL3- and RPL3L-containing ribosomes modulates mitochondrial activity in the mammalian heart. Nucleic Acids Res. 2023;51(11):5301-24.</li>
	<li>Sloan KE, et al. Tuning the ribosome: the influence of rRNA modification on eukaryotic ribosome biogenesis and function. RNA Biol. 2017;14(9):1138-52.</li>
	<li>Simsek D, Barna M. An emerging role for the ribosome as a nexus for post-translational modifications. Curr Opin Cell Biol. 2017;45:92-101.</li>
	<li>Baghirova S, Hughes BG, Hendzel MJ, Schulz R. Sequential fractionation and isolation of subcellular proteins from tissue or cultured cells. MethodsX. 2015;2:440-5.</li>
	<li>Jagannathan S, Nwosu C, Nicchitta CV. Analyzing mRNA localization to the endoplasmic reticulum via cell fractionation. Methods Mol Biol. 2011;714:301-21.</li>
	<li>Fairhead M, Howarth M. Site-specific biotinylation of purified proteins using BirA. Methods Mol Biol. 2015;1266:171-84.</li>
	<li>Jan CH, Williams CC, Weissman JS. Principles of ER cotranslational translocation revealed by proximity-specific ribosome profiling. Science. 2014;346(6210):1257521.</li>
	<li>Dakshinamurti K, Chalifour L, Bhullar RP. Requirement for biotin and the function of biotin in cells in culture. Ann N Y Acad Sci. 1985;447:38-55.</li>
	<li>Roux KJ, Kim DI, Raida M, Burke B. A promiscuous biotin ligase fusion protein identifies proximal and interacting proteins in mammalian cells. J Cell Biol. 2012;196(6):801-10.</li>
	<li>Branon TC, et al. Efficient proximity labeling in living cells and organisms with TurboID. Nat Biotechnol. 2018;36(9):880-7.</li>
	<li>Rhee HW, et al. Proteomic mapping of mitochondria in living cells via spatially restricted enzymatic tagging. Science. 2013;339(6125):1328-31.</li>
	<li>Benedetti L, et al. Optimized Vivid-derived Magnets photodimerizers for subcellular optogenetics in mammalian cells. Elife. 2020;9:e63230.</li>
	<li>Zhang Z, et al. A subcellular map of translational machinery composition and regulation at the single-molecule level. Science. 2025;387(6738):eadn2623.</li>
	<li>Weinberg BH, et al. High-performance chemical- and light-inducible recombinases in mammalian cells and mice. Nat Commun. 2019;10(1):4845.</li>
	<li>Goldman A, Harper S, Speicher DW. Detection of proteins on blot membranes. Curr Protoc Protein Sci. 2016;86:10.8.1-10.8.11.</li>
	<li>Gravel P. Protein blotting by the semidry method. In: Walker JM, editor. The protein protocols handbook. 2nd ed. Totowa, NJ: Humana Press; 2002. p. 321-34.</li>
	<li>Kirkeby S, Moe D, Bog-Hansen TC, Van Noorden CJ. Biotin carboxylases in mitochondria and the cytosol from skeletal and cardiac muscle as detected by avidin binding. Histochemistry. 1993;100(6):415-21.</li>
	<li>Chandler CS, Ballard FJ. Multiple biotin-containing proteins in 3T3-L1 cells. Biochem J. 1986;237(1):123-30.</li>
	<li>Jaaro H, Rubinfeld H, Hanoch T, Seger R. Nuclear translocation of mitogen-activated protein kinase kinase (MEK1) in response to mitogenic stimulation. Proc Natl Acad Sci U S A. 1997;94(8):3742-7.</li>
	<li>Dang CV, Lee WM. Identification of the human c-myc protein nuclear translocation signal. Mol Cell Biol. 1988;8(10):4048-54.</li>
	<li>Snaar S, et al. Mutational analysis of fibrillarin and its mobility in living human cells. J Cell Biol. 2000;151(3):653-62.</li>
	<li>Shibata Y, et al. The reticulon and DP1/Yop1p proteins form immobile oligomers in the tubular endoplasmic reticulum. J Biol Chem. 2008;283(27):18892-904.</li>
	<li>Palmer AE, et al. Ca2+ indicators based on computationally redesigned calmodulin-peptide pairs. Chem Biol. 2006;13(5):521-30.</li>
	<li>Seth RB, Sun L, Ea CK, Chen ZJ. Identification and characterization of MAVS, a mitochondrial antiviral signaling protein that activates NF-kappaB and IRF3. Cell. 2005;122(5):669-82.</li>
	<li>Collins JC, et al. Biotin-dependent expression of the asialoglycoprotein receptor in HepG2. J Biol Chem. 1988;263(23):11280-3.</li>
	<li>Moskowitz M, Cheng DK, Moscatello DK, Otsuka H. Growth stimulation of BHK cells in culture by free biotin in serum. J Natl Cancer Inst. 1980;64(3):539-44.</li>
</ol>

```html
<table><tbody><tr><td>-80 &deg;C storage</td><td></td><td></td><td>細胞培養用</td></tr><tr><td>0.1% gelatin in water</td><td>MilliporeSigma</td><td>ES-006-B</td><td>細胞培養用</td></tr><tr><td>0.2 &micro;m filter units</td><td>Fisher</td><td>04-977-158</td><td>細胞培養用</td></tr><tr><td>0.5% trypsin-EDTA (10X)</td><td>ThermoFisher</td><td>15400-054</td><td>細胞培養用</td></tr><tr><td>1 M MgCl2</td><td>ThermoFisher</td><td>AM9010</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>1 M Tris pH 7.5</td><td>ThermoFisher</td><td>15567027</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>1 M Tris pH 8</td><td>ThermoFisher</td><td>AM9855G</td><td>オプション：エキソヌクレアーゼタグ付きタンパク質をリボソームタンパク質の内因性遺伝子座に挿入する場合</td></tr><tr><td>1.5 and 2 mL microcentrifuge tubes, autoclaved</td><td></td><td></td><td>トランスフェクション用</td></tr><tr><td>1.5, 2 mL, and 5 mL protein low-binding microcentrifuge tubes</td><td>Eppendorf</td><td>022431081, 022431102, 0030108302</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>10 cm tissue culture plates</td><td></td><td></td><td>細胞培養用</td></tr><tr><td>10 M NaOH</td><td>MilliporeSigma</td><td>72068-100ML</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>10x Tris/glycine/SDS running buffer concentrate</td><td>Bio-Rad</td><td>1610772</td><td>ウェスタンブロット用</td></tr><tr><td>37 &deg;C tissue culture incubator x 2</td><td></td><td></td><td>細胞培養用。1台は主要な細胞培養インキュベーターおよびトランスフェクト細胞の照明用として機能し、2台目はALIBi活性化日のテンポラリスペースとして、主インキュベーター内のトランシリミネータのセットアップ中にトランスフェクト細胞を収容する。</td></tr><tr><td>37 &deg;C water bath</td><td></td><td></td><td>細胞培養用</td></tr><tr><td>5 M NaCl</td><td>ThermoFisher</td><td>AM9010</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>50 mM biotin</td><td>ThermoFisher</td><td>B20656</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>6x Laemmli buffer</td><td>FIsher</td><td>50-196-784</td><td>ウェスタンブロット用</td></tr><tr><td>70% ethanol</td><td></td><td></td><td>消毒用</td></tr><tr><td>Acetonitrile</td><td>Fisher</td><td>A955</td><td>オプション：マススペクトルサンプル調製用</td></tr><tr><td>AcTEV protease kit</td><td>ThermoFisher</td><td>12575015</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>ALIBi C-fragment plasmid</td><td>Addgene</td><td>#235615, 235636 &ndash; 235641</td><td>トランスフェクション用にはMidi-prepスケールが必要</td></tr><tr><td>ALIBi N-fragment plasmid</td><td>Addgene</td><td>#235614, 235635, 235642, 235643</td><td>トランスフェクション用にはMidi-prepスケールが必要</td></tr><tr><td>Aluminum foil</td><td></td><td></td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>ammonium bicarbonate</td><td>MilliporeSigma</td><td>5.3305</td><td>オプション：マススペクトルサンプル調製用</td></tr><tr><td>anti-FLAG antibody</td><td>MilliporeSigma</td><td>F3165</td><td>ウェスタンブロット用</td></tr><tr><td>anti-GAPDH antibody</td><td>Cell Signaling</td><td>5174</td><td>ウェスタンブロット用</td></tr><tr><td>anti-mouse secondary antibody</td><td>GE Healthcare</td><td>NA931V</td><td>ウェスタンブロット用</td></tr><tr><td>anti-rabbit secondary antibody</td><td>GE Healthcare</td><td>NA934V</td><td>ウェスタンブロット用</td></tr><tr><td>anti-Rpl12 antibody</td><td>Abcam</td><td>ab175219</td><td>ウェスタンブロット用</td></tr><tr><td>anti-Rpl31 antibody</td><td>Abcam</td><td>ab103991</td><td>ウェスタンブロット用</td></tr><tr><td>anti-Rpl5 antibody</td><td>ProteinTech</td><td>15430-1-AP</td><td>ウェスタンブロット用</td></tr><tr><td>anti-V5 antibody</td><td>ThermoFisher</td><td>R960-25</td><td>ウェスタンブロット用</td></tr><tr><td>Apyrase</td><td>NEB</td><td>M0398L</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>Beta-mercaptoethanol (55 mM)</td><td>ThermoFisher</td><td>21985-023</td><td>細胞培養用</td></tr><tr><td>Blue light transilluminator</td><td>Clare Research Chemical</td><td>DR46B (small) or DR196 (large)</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>Bovine serum albumin (BSA)</td><td>Fisher</td><td>BP1600-100</td><td>ウェスタンブロット用</td></tr><tr><td>C18 tips (OMIX C18 tips)</td><td>Agilent</td><td>A57003100</td><td>オプション：マススペクトルサンプル調製用</td></tr><tr><td>Cell freezing container</td><td></td><td></td><td>細胞培養用</td></tr><tr><td>Centrifuge fitted for 15 mL and/or 50 mL conical tubes</td><td></td><td></td><td>細胞培養用</td></tr><tr><td>Centrifuge for 1.5-2 mL microcentrifuge tubes</td><td></td><td></td><td>溶解 &amp; アフィニティ精製用。4 Cまで冷却可能、17,000 gまでの速度が可能なこと。理想的には、チューブを出し入れする際に一時的に暗くできる部屋に配置されていること。</td></tr><tr><td>Clarity ECL kit</td><td>Bio-Rad</td><td>170-5061</td><td>ウェスタンブロット用</td></tr><tr><td>Criterion gel tank</td><td>Bio-Rad</td><td>1656001</td><td>ウェスタンブロット用</td></tr><tr><td>Criterion TGX pre-cast gels, 4-20%</td><td>Bio-Rad</td><td>5671095</td><td>ウェスタンブロット用。追加のウェルサイズとレイアウトが利用可能</td></tr><tr><td>Cycloheximide</td><td>MilliporeSigma</td><td>C7698-5G</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>Deoxycholate</td><td>MilliporeSigma</td><td>S1827</td><td>溶解 &amp; アフィニティ精製用</td></tr><tr><td>Dithiothreitol</td><td>ThermoFisher</td><td>PIA39255</td><td>オプション：マススペクトルサンプル調製用</td></tr><tr><td>DPBS, no calcium, no magnesium</td><td>ThermoFisher</td><td>14190-250</td><td>細胞培養用</td></tr><tr><td>ES-qualified FBS</td><td>MilliporeSigma</td><td>ES-009-B</td><td>細胞培養用</td></tr><tr><td>Ethanol</td><td>Gold Shield</td><td>412828</td><td>ウェスタンブロット用</td></tr><tr><td>Ethanol</td><td>MilliporeSigma</td><td>459828</td><td>オプション：マススペクトルサンプル調製用</td></tr><tr><td>Formic acid</td><td>MilliporeSigma</td><td>A117</td><td>オプション：マススペクトルサンプル調製用</td></tr><tr><td>Fragment size analyzer and consumables</td><td></td><td>Agilent</td><td>オプション：RNAシーケンスライブラリ調製用。Agilent 2100 Bioanalyzer</td></tr><tr><td>Freezing media</td><td>ThermoFisher</td><td>12648-010</td><td>細胞培養用</td></tr><tr><td>

組換えペプチドタグ特異的位置制限照射強化ビオチニル化に基づく細胞内リボソームサブポピュレーションの単離

Isolation of Subcellular Ribosome Subpopulations Based on Recombinant Peptide Tag-Specific Location-Restricted Illumination-Enhanced Biotinylation

Growing evidence suggests that mRNA translation is a highly compartmentalized process within a cell, and that subcellular trafficking and localization of ...

Our research investigates how ribosome composition and mRNA translation vary across subcellular domains to regulate local protein synthesis. Current methods lack spatial and temporal precision. ALIBi addresses this through optogenetically controlled proximity labeling of ribosomes.To begin, obtain L31FTA mouse embryonic stem cells and transfect them with ALIBi, GFP control, or full length BirA plasmids. After 24 to 48 hours of transfection, pre-chill the centrifuge to four degrees Celsius. With the lights off, move the transfected plates to a temporary 37-degree Celsius incubator.To prevent temperature fluctuations, once the transilluminator is placed in the main incubator, using 70%ethanol, wipe down the blue light transilluminator. After connecting the transilluminator to power, place it in the main incubator. Allow the incubator conditions to reequilibrate, while continuing the setup.Warm an aliquot of media to 37 degrees Celsius for dosing with biotin and cyclohexamide, or CHX. If preparing control samples without supplemental biotin, warm a separate aliquot for CHX-only dosing. Chill an aliquot of DPBS intended for CHX dosing.Prepare the RNase removal buffer, salt wash, lysis buffer, and wash buffers. Using a pipette, mix buffers containing dense or viscous components to ensure even mixing. Chill the prepared buffers on ice before use.Re-suspend the stock bottle of streptavidin beads by gentle pipetting or rocking. Transfer 450 microliters of beads into a protein low binding tube for each 10 centimeter plate of cells. Place the re-suspended beads on the magnetic rack for one minute, or until the beads have fully migrated.Aspirate and discard the bead storage buffer without touching the bead pellet. Wash the beads two times with 900 microliters of RNAs removal buffer. Wash the beads once with 900 microliters of salt wash.Wash the beads two times with 900 microliters of wash I buffer. Store the beads in the wash I buffer at four degrees Celsius while performing activation and cell lysis. Add biotin and CHX to the warmed aliquot of media and keep at 37 degrees Celsius until use.Add CHX to the cold DPBS aliquot and keep it chilled until use. Aspirate the media from the transfected plates. Immediately add 10 milliliters of warm media containing CHX with or without Biotin to each plate.Place negative control plates in a dark incubator. Place the remaining plates directly on top of the transilluminator. Turn on the transilluminator and incubate the plates for 15 minutes.Wash each plate two times with the 10 milliliters of cold DPBS containing CHX. Place the plates on ice between and after washes. After aspirating the second wash, tilt the plate and aspirate any residual DPBS.Keeping the plate flat on ice, add two milliliters of ice-cold lysis buffer and incubate the plate on ice for five minutes. Tap the plate two to three times during incubation to dislodge cells. Using a micropipette, pipette the lysate up and down across the plate surface to dislodge any remaining cells.Transfer the lysate to a chilled labeled protein low binding tube. In difficult to lyse organelles, sonicate all samples for 45 seconds at four degrees Celsius. And if required, transfer the lysate into a larger tube before sonication.Transfer the sonicated lysate into a two-milliliter tube. Incubate the lysate on ice for 15 minutes. Centrifuge the lysate at 17, 000 G for three minutes at four degrees Celsius.Transfer the supernatant into a chilled protein low binding tube without disturbing the pellet. Using a magnetic rack, pellet the washed strepped avident beads. Aspirate the wash one buffer.Transfer 1, 200 microliters of clarified lysate to the tube containing the washed beads. Pipette up and down gently to re-suspend the beads. Store the remaining clarified lysate at minus 80 degrees Celsius.Incubate the lysate with the beads at four degrees Celsius in the dark on a tube rotator for one hour. During the incubation, prepare the Tobacco Etch Virus, or TEV elution buffer. Pellet the beads on a magnetic rack.Transfer the supernatant to a chilled protein low binding tube. Store the supernatant at minus 80 degrees Celsius. Wash the beads two times with 900 microliters of wash I buffer and once with 900 microliters of wash II buffer.Add 450 microliters of TEV elution buffer to the beads. Incubate the beads in TEV elution buffer for one hour at room temperature on a tube rotator. Pellet the beads on the magnetic rack.Transfer the elute to a protein low binding tube. Store the eluate at minus 80 degrees Celsius if proteomics analysis or Western blot is intended. Cells containing all system components showed a L31-FTA band, which was stronger with full length burray and faint or absent when components were omitted.Endogenous biotinilated carboxylase proteins were detected in all lysates at 70 to 80 kilodaltons and 120 to 130 kilodaltons. ANTV5 probing confirmed expression of the split bore fragments. Biotinillated L31FTA decreased in the flow through relative to the lysate.No biotinillated L31-FTA was present in the eluate following TEV-mediated elution. Anti-flag probing showed a cleaved L31-FTA band at a lower molecular weight than intact L31-FTA ribosomal protein. S12 probing demonstrated the presence of the small ribosomal subunit in the eluate.Anti-V5 probing showed that proteins that do not bind the ribosome were absent from the eluate. When 2.5 fold more lysate was used with the same 450 microliters of streptavidin coated beads, a substantial amount of biotinilated L31-FTA remained in the flow through. Endogenous biotinilated proteins were partially depleted in the flow through.Endogenous biotinillated proteins were absent from the eluet, but were recovered after heating post-dilution beads in lambly buffer. Anti-flag probing detected cleaved L31-FTA at a lower molecular weight than intact L31-FTA. Anti-V5 probing showed that the split burray fragments were absent from the eluate.ALIBi enables the identification of ribosome-bound proteins and mRNAs in specific subcellular regions, revealing differences in translation regulation. The key challenge is maintaining specific proximity labeling through careful control of targeting construct expression and illumination conditions. Following this protocol, affinity purified ribosomes and associated mRNAs can be analyzed using quantitative proteomic and transcriptomic approaches.

isolation-subcellular-ribosome-subpopulations-based-on-recombinant

Research

JoVE Journal

Biology

生物学

ペプチジル- tRNAの初期段階を含むリボソームの生化学的解析への大きな障害は、同じサンプル中の他のリボソーム、解析対象の特定のmRNA配列の翻訳に関与していないリボソームの存在となっています。我々は、排他的に、興味の発生期のペプチジル-...

機能と構造解析のためにペプチジル- tRNAを含む翻訳リボソームの分離

mRNA上の翻訳停止部位を同定する技術が記載されている。この手順は、変性ゲル電気泳動を用いた初期のチェーンの大きさの分析に続いて標的mRNAのin...

リボソーム結合した新生ポリペプチドの分離<em&gt; in vitroで</em&gt; mRNAに沿って並進一時サイトを識別するために

ここでは今定期的に安定的に結合したリボソーム新生鎖複合体（のRNC）を分離するために使用される手法について説明します。この手法は、17アミノ酸長いSECM...

リボソーム結合したポリペプチドを単離するためのツールと​​してSECM逮捕シーケンスを使用して、

機能的モチーフとその結合パートナーのペプチドベース識別

このプロトコルは、in vivoでのCNSサンプルのtranslatomeを研究するためにポリリボソーム分画の本質的な変更を示している。これは、結合したRNA画分をリボソームへの全RNAの単離と比較を通じて翻訳と転写制御の全体的な評価が可能になります。...

ポリリボソーム画によって中枢神経系Translatomeの生体尋問中

ここでは、光親和性標識を使用して低分子結合タンパク質を同定する方法を説明します。この技術の利点は、標的タンパク質の結合および共有結合標識が生細胞環境内で行われるため、細胞溶解時に天然タンパク質の構造および結合条件を破壊するリスクが排除されることです。...

ライブセル光親和性標識によって、ネイティブの細胞環境における小分子結合タンパク質の同定

複雑な生体試料から生じるユビキチン化タンパク質を起源とするdiGlyペプチドの精製、検出、同定方法を紹介します。提示された方法は、ユビキチノメ分析の深さのレベルに関して、再現性、堅牢性、および優れたメソッドを上回る。...

ペプチド濃縮と質量分析によるタンパク質ユビキチン化部位の検出

ここでは、リボタグシステムを用いた成人男性マウス生殖細胞の選択集団からリボソームおよび関連RNAの免疫沈降について説明する。戦略的な繁殖と慎重な免疫沈降は、細菌細胞の翻訳を知らせ、変異型のメカニズムに関する洞察を提供するクリーンで再現可能な結果をもたらします。...

雄マウスの生殖細胞におけるリボタグ免疫沈降

単一分子蛍光エネルギー移動は、リボソームタンパク質合成中のtRNAダイナミクスを追跡する方法である。個々のリボソームを追跡することにより、不均一な集団が同定され、メカニズムに光が当たります。この方法は、生物学的立体構造の変化を一般的に追跡し、他の多くの複雑なバイオシステムにおける動的機能関係を明らかにするために使用することができる。単一分子法は、平均効果のために従来のアンサンブル法ではアクセスできない非レート制限ステップおよび低人口のキー中間体を観察することができる。...

リボソームタンパク質合成の単一分子蛍光エネルギー転移研究

共翻訳相互作用は、新生鎖修飾、標的化、折り畳み、およびアセンブリ経路において重要な役割を果たします。ここで、選択的リボソームプロファイリング、 in vivo、モデル真核生物...

選択的リボソームプロファイリングによる共翻訳相互作用ネットワークのグローバル同定

プロテオームスケールでのタンパク質の発見と定量に適合する高品質のミトコンドリア単離のための2段階プロトコルを提示します。私たちのプロトコルは遺伝子工学を必要としないため、あらゆる初代細胞や組織からのミトコンドリアの研究に適しています。...

タンパク質の発見と定量を改善するためのミトコンドリアの2段階タグフリー分離

Stanford School of Medicine.  AviTag特異的位置制限照明強化ビオチニル化(ALIBi)は、リボソームの細胞内集団を標識し親和性を精製し、下流のシーケンスおよびプロテオーム解析のために開発された新しいオプトジェネティック活性化法です。

組換えペプチドタグ特異的位置制限照射強化ビオチニル化に基づく細胞内リボソームサブポピュレーションの単離

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