Name: desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells
Uploaded: 2018-04-25T15:00:00
Description: Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

この作品は、スイス国立科学財団 Sinergia グラント CRSII3 147697、財団毛皮アメリカ Krebsforschung、チューリッヒ Krebsliga によって支えられました。

1. ゾル性細胞質、核タンパク質画分の調製
<ol><li>プレート 4 × 106 ACC-メソ-4 RPMI - 1640 媒体で育つ T150 細胞培養用フラスコ細胞 10 %fbs、1 x ペニシリン/ストレプトマイシン (100 x) 2 mM L-グルタミン (200 mM) と補われます。細胞が 80-90% (~5-5.5 x 106セル) の合流点に達したら、核と細胞質画分の蛋白質の抽出を続行します。 注: ACC-メソ-4 セルの行は、理化学研究所バイオ リソース セ...

<ol>
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3' UTRs は非コーディング ゲノム3に属し、すべての非コード Rna 蛋白質7彼らの機能を発揮するために対話できます。哺乳類のゲノムは行き渡って転写されることがわかったし、長鎖非コード Rna16のかなりの部分を生成、これらの長い非コード Rna 機能と対話する遺伝子発現を調節することを示した証拠を新興クロマチン再構築複合体<sup class...

MRNA の安定性と mRNA 翻訳レート1に影響を与える遺伝子の発現と遺伝子産物の最後のレベルをしっかりと調節できます。非コーディングの RNA や RNA 結合タンパク質 (Rbp) の標的 mRNA との対話を通じてこれらの転写後調節機構が加わる。それは通常 (3' UTR -2ゲノムの非コーディングの部分に属する) 特定cisを含む mRNA の 3' 非翻訳領域-トランスによって認識される規制要素 (CRE)-miRNA や Rbp などの要因を演技3最高研究cis-3' UTR、内の要素は、アデニンの豊富な要素 (は) モチーフには、特定の AU 結合蛋白質 (AUBP) し、順番が認識誘導か mRNA の分解/deadenylation (は崩壊) または。mRNA 安定4。
サイズ 3' UTR カルレチニンの mRNA (CALB2) は 573 長い bp、AREsite2、bioinformatic ツール5によって予測された、推定 AUUUA いくつが含まれます。推定はモチーフの存在と一貫性のある、pmirGLO ベクトル レポーターアッセイ CALB2 mRNA6要素の安定化の役割を示した。最後にの in vitro RNA プルダウン カルレチニンが安定 AUBP を識別するために使用された mRNA はモチーフを。
すべての非コーディングの Rna とタンパク質7の対話、以来体外RNA プルダウンは分子メカニズムの解読を支援するための良い方法と RNA インターアクターを識別するための最初の選択の試金。この RNA プルダウン法市販合成 RNA プローブは、RNA 鎖の 3' 末端をビオチン (desthiobiotin) の修正された形で付けられましたが使用されていました。RNA desthiobiotin が特に関心のバインド RNA と相互作用するタンパク質をプルダウンに使用される磁気ビーズのストレプトアビジンとの相互作用を介して固定します。中皮腫細胞の細胞質画分から非変性とアクティブな蛋白質は蛋白質のソースとして使用されます。このような RNA 結合タンパク質は、12 %sds ページのゲルを実行、膜に転送され、異なる抗体をプローブの磁気ビーズから溶出されます。
過酷な変性条件は8バインドされたタンパク質を溶出への強力な不可逆的なビオチン ストレプトアビジン結合を破壊するために必要な標準的なストレプトアビジン-ビオチン アフィニティ精製手順での解離を引き起こす可能性蛋白質の複合体。異なり、ビオチン、desthiobiotin は可逆的ストレプトアビジンにバインド、ビオチン、蛋白質の穏やかな溶出を可能にして当然のことながらビオチン化分子の9分離を回避するバッファー溶液で避難は競争力のあります。技法が原産の条件の下でネイティブ蛋白質の複合体を分離するために理想的なことを示唆しています。
体外結合条件と逼迫、塩濃度、還元剤、洗剤の割合によって決定されますが真生体内での相互作用を識別するために携帯電話のコンテキストで現在のものに近いはずです。本実装バインド条件実証されている以前 AU 結合タンパク質10として、ハーを識別するために適切です。このアプローチは、適切な結合条件の最適化は時間がかかり、挑戦をすることができますので、時間を節約できます。さらに、このメソッドは、任意の RNA プルダウン実験開始プロトコルとして使用することができる、塩と洗剤の濃度を変化させ、グリセリンの割合を変更して他の塩を追加することによって徐々 に最適化することができます。さらに、こともいくつをかくまっている短い 25 ヌクレオチド RNA プローブはモチーフに使える特定の AUBP との相互作用を示すデモンストレーションを行った。

<table border="0" cellpadding="0" cellspacing="0" width="1340">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">NE-PER Nuclear and cytoplasmic extraction kit</td>
			<td style="width:295px;">Thermo Fisher Scientific&#160;</td>
			<td style="width:124px;">78833</td>
			<td style="width:468px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Pierce Protease Inhibitor Tablets, EDTA-free</td>
			<td>Thermo Fisher Scientific</td>
			<td style="width:124px;">A32965</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce BCA Protein Assay Kit</td>
			<td>Thermo Fisher Scientific</td>
			<td>23225</td>
			<td></td>
		</tr>
		<tr height="24">
			<td height="24" style="height:24px;">Pierce Magnetic RNA-protein Pull-Down Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20164</td>
			<td>Includes magnetic beads and therefore the kit need to be stored at 4 &#176;C</td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;">Pierce RNA 3&#8217; End Desthiobiotinylation Kit&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">20163</td>
			<td style="width:468px;">The kit is a part of the &#34;Pierce Magnetic RNA-protein Pull-Down Kit&#34;, and needs to be stored at -20 &#176;C unlike the pull-down kit (4 &#176;C).</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">DynaMag-2,&#160; magnetic stand</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">12321D</td>
			<td></td>
		</tr>
		<tr height="63">
			<td height="63" style="height:63px;width:453px;">Anti - HuR (MOUSE) monoclonal antibody</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">-</td>
			<td style="width:468px;">The antibody is included in the Pierce Magnetic RNA-protein Pull-Down Kit&#160; - 1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - &#945; - tubulin (MOUSE) monoclonal antibody</td>
			<td>Santa Cruz</td>
			<td style="width:124px;">8035</td>
			<td>1:1000 in 5% BSA 1x TTBS - rabbit anti-mouse secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - PARP (RABBIT) Polyclonal antibody</td>
			<td>Cell Signaling</td>
			<td>9542</td>
			<td>1:1000 in 5% BSA 1x TTBS - goat anti-rabbit secondary antibody 1:10,000 in 5% BSA 1x TTBS</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Anti - Mesothelin (MOUSE) Monoclonal Antibody&#160;</td>
			<td>Rockland</td>
			<td>200-301-A87</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary goat anti-rabbit antibody</td>
			<td style="width:295px;">Cell Signaling</td>
			<td style="width:124px;">7074</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Secondary rabbit anti-mouse antibody</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A-9044</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RPMI - 1640 medium</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td>R8758</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FBS - Filtrated Bovine Serum</td>
			<td style="width:295px;">Pan Biotech&#160;</td>
			<td style="width:124px;">P40-37500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Penicillin - streptomycin (100x)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">P4333</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">L-Glutamine solution (200 mM)</td>
			<td style="width:295px;">Sigma-Aldrich&#160;</td>
			<td style="width:124px;">G7513</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">0.25% Trypsin-EDTA (1x)</td>
			<td style="width:295px;">Gibco by Life technologies&#160;</td>
			<td style="width:124px;">25200-056</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN 3 Cell</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653301</td>
			<td></td>
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		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini Protean System Glass Plates&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653308</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Spacer Plates with 1.5 mm integrated spacer</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653312</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Mini-PROTEAN Comb, 10-well, 1.5 mm, 66 &#181;L</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1653365</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Mini-PROTEAN Tetra Cell Casting Modules</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1658050</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNaseZap RNase Decontamination Solution</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">AM9780</td>
			<td></td>
		</tr>
		<tr height="42">
			<td height="42" style="height:42px;width:453px;">Rotiphorese gel 30 - aqueous 30% acrylamide and bisacrylamide stock solution at a ration of 37.5:1</td>
			<td style="width:295px;">Roth</td>
			<td style="width:124px;">3029</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">20% SDS&#160;</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td>A3942</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">PVDF transfer membrane&#160;</td>
			<td style="width:295px;">Perkin Elmer</td>
			<td style="width:124px;">NEF1002</td>
			<td>Need to be activated by incubating in pure methanol for 1 min followed by washing in water for 2 min&#160;</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Trans-Blot SD semi-dry transfer cell&#160;</td>
			<td style="width:295px;">Bio-Rad</td>
			<td>1703940</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Clarity Western ECL Substrate</td>
			<td style="width:295px;">Bio-Rad</td>
			<td style="width:124px;">1705061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">FusionFX&#160; Digital Imager</td>
			<td style="width:295px;">Vilber</td>
			<td style="width:124px;">-</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">RNA oligo synthesis</td>
			<td style="width:295px;">Mycrosynth AG, Switzerland</td>
			<td style="width:124px;">-</td>
			<td>the synthesis scale - 0.04 &#181;mol - HPLC purified</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Bovine serum albumin</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A7030</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Hydrochloric Acid (HCl) 6 M</td>
			<td style="width:295px;">PanReac AppliChem</td>
			<td style="width:124px;">182883.1211</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ultra Pure 1 M Tris, pH 7.5&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">15567027</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Glycerol&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td>17904</td>
			<td>50% sterile aliquots to be stored at -20&#176;C</td>
		</tr>
		<tr height="105">
			<td height="105" style="height:105px;width:453px;">Pierce Streptavidin magnetic beads&#160;</td>
			<td style="width:295px;">Thermo Fisher Scientific</td>
			<td style="width:124px;">88817</td>
			<td style="width:468px;">Please note that these magnetic beads have an average diameter of 1 &#181;m (range from 0.5 - 1.5 &#181;m). Furthermore, Dynabeads-M280 Streptavidin, which are beads of homogenouse size 2.8 &#181;m, are also used in RNA-pulldown but be aware that this may affect purification process.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">TEMED</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">T9281</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Ammonium persulfate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">A3678</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:453px;">Coomassie G-250</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A3480</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Aluminiumsulfate-(14-18)-hydrate&#160;</td>
			<td style="width:295px;">Sigma-Aldrich</td>
			<td style="width:124px;">368458</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">ortho-phosphoric acid&#160;</td>
			<td style="width:295px;">Applichem</td>
			<td style="width:124px;">A0637</td>
			<td></td>
		</tr>
	</tbody>
</table>

desthiobiotin-streptavidin-affinity mediated purification of rna-interacting proteins in mesothelioma cells

In vitro RNA プルダウン、まだ主目的で特定の RNA の構造とモチーフを認識 RNA 結合蛋白質を識別するプロトコルの最初の手順で使用されます。この RNA プルダウン プロトコルで市販合成 RNA プローブがビオチン、ストレプトアビジンに可逆的に結合しより下で蛋白質の溶出がように rna の 3' 末端に、desthiobiotin の変更されたフォームと分類される生理条件。RNA desthiobiotin が特に関心の RNA と相互作用するタンパク質をプルダウンに使用される磁気ビーズのストレプトアビジンとの相互作用を介して固定します。中皮腫細胞の細胞質画分から非変性とアクティブな蛋白質は蛋白質のソースとして使用されます。ここで説明する方法は、知られている RNA 結合タンパク質との興味のシーケンスを含む 25 ヌクレオチド (nt) 長い RNA プローブ間の相互作用の検出に適用できます。これは安定しているか不安定要素ベクトル レポーターアッセイを使用して達成する RNA 分子の存在の機能の解析を完了するのに役立ちます。

この実験では、カルレチニン 3' UTR 遺伝子がモチーフ (CALB2 3' UTR (は) 25 nt) の 25 nt 長いフラグメントはそれは既知 mRNA 安定人間抗原 R (ハー) 蛋白質にとりわけ結合するかどうかをテストするため使用されました。25 nt RNA は要素の特異性をテストするには、プローブ CALB2 3' UTR (mtARE) がモチーフの安定化効果を廃止する以前に示されて、あるモチーフの突然変異を含むだ?...

Watch this Scientific Journal Video about Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells at JoVE.com

Desthiobiotin-Streptavidin-Affinity Mediated Purification of RNA-Interacting Proteins in Mesothelioma Cells

Desthiobiotin ストレプトアビジンの親和性による中皮腫細胞の RNA 結合タンパク質の精製

Desthiobiotin ラベル アデニンの豊富な要素 (は) モチーフが含まれている、合成 25 ヌクレオチド RNA oligo のゾル性細胞質が結合タンパク質の特異的結合をことができます。

The in vitro RNA-pulldown is still largely used in the first steps of protocols aimed at identifying RNA-binding proteins that recognize specific RNA ...

The overall goal of this procedure is to capture cytosolic protein that bind to a specific sequence located in RNA molecule in mesothelioma cells. This method can help answer key question in RNA biology field, such as if a predicted RNA binding protein can bind adenosine or uridine-rich sequence in a transcript. The main advantage of this technique is that it complements functional characterization of stabilizing or destabilizing elements assessed by reporter assay.Demonstrating this technique will be Dr.Jelena Kresoja-Rakic, a postdoc from my laboratory. To begin the protocol, obtain previously prepared ACC-MESO-4 cells with a 80 to 90%confluence. Aspirate the medium, wash the cells with 15 milliliters of 1X phosphate buffered saline, or PBS, and aspirate the PBS.Next, add three milliliters of 0.25%trypsin-EDTA, and incubate the culture flask at 37 degrees Celsius for three minutes. Add seven milliliters of supplemented RPMI-1640 medium, collect the cells in a 15-milliliter tube, and spin them down at 200 times g for five minutes. Then, aspirate the medium, and resuspend the cell pellet with 1.5 milliliters of 1X PBS.Then, transfer the cells into a 1.5-milliliter microcentrifuge tube. Spin down the cells again at 500 times g and four degrees Celsius for five minutes. After the spin, discard the supernatant, resuspend the cell pellet with 1.5 milliliters of ice-cold 1X PBS, and spin down the cells at 500 times g and four degrees Celsius for three minutes.Discard the supernatant, and estimate the cell pellet packed volume by comparing the tube containing the cell pellet with a 1.5-milliliter tube with 10, 20, 50, and 100 microliters of 1X PBS. Add 200 microliters of ice-cold cytoplasmic extraction reagent, or CER I, supplemented with two microliters of 1X proteinase inhibitor. Vigorously vortex the pellet for 15 seconds, and incubate the tube on ice for 10 minutes.Add 11 microliters of ice-cold cytoplasmic extraction reagent II, or CER II, vigorously vortex for five seconds, and incubate the tube for one minute on ice. Vortex the sample vigorously for five seconds, and centrifuge the tube at 16, 000 times g for five minutes. Transfer the supernatant to the clean, pre-chilled tube on ice.Resuspend the remaining pellet in 100 microliters of ice-cold nuclear extraction reagent, or NER, supplemented with one microliter of proteinase inhibitor, and vigorously vortex for 15 seconds. Incubate the sample on ice for 40 minutes, and vortex every 10 minutes for 15 seconds. Centrifuge the tube at 16, 000 times g for 10 minutes.Transfer the supernatant, which is the nuclear protein fraction, to a clean, pre-chilled tube. Immediately measure the protein concentration using the bicinchoninic method. Then, prepare 25 microliters of aliquots of cytosolic and nuclear extracts.Snap-freeze these aliquots by placing them in liquid nitrogen for five seconds, and store them directly at minus 80 degrees Celsius. Transfer five microliters of 10-micromolar RNA probes into 0.5-milliliter, thin-wall microcentrifuge tubes, and incubate them in a PCR machine at 85 degrees Celsius for five minutes. Place the tubes immediately on ice.For a single 30-microliter reaction, add 10 microliters of previously prepared mix to the RNA-containing tube. Carefully add 15 microliters of PEG 30%to the reaction, and use a fresh tip to mix the reaction. Incubate the reaction overnight at 16 degrees Celsius.The next day, prepare the reagents. Add 70 microliters of nuclease-free water to the RNA-labeling reaction tubes. Add 100 microliters of chloroform-isoamyl alcohol, and vortex briefly and spin the sample at 13, 000 times g for three minutes.Carefully remove only the upper phase. Avoid touching the lower phase. Transfer it to a new nuclease-free, 1.5-milliliter tube.Add 10 microliters of five-molar sodium chloride, one microliter of glycogen, and 300 microliters of ice-cold 100%ethanol. Place the tube at minus 20 degrees Celsius for two hours. After two hours, centrifuge at 13, 000 times g and four degrees Celsius for 15 minutes.Carefully discard the supernatant without disturbing the pellet. Add 300 microliters of ice-cold 70%ethanol, and centrifuge again at 13, 000 times g and four degrees Celsius for five minutes. Discard the supernatant completely, and air-dry the pellet for 15 minutes.Resuspend the pellet in 20 microliters of nuclease-free water. Incubate the RNA at 90 degrees Celsius for two minutes, and place it on ice. Place the labeled RNA on ice during the following step of pre-washing of the streptavidin magnetic beads.Prewash 50 microliters of streptavidin magnetic beads per 50 picomoles of RNA. Vortex the tube with streptavidin magnetic beads for 15 seconds. After vortexing quickly, remove 200 microliters into a clean, 1.5-milliliter Safe-Lock tube using cut pipette tips.Next, place the tube on a magnetic stand so that the beads collect at the side of the tube, and wait one minute. Remove the resuspension liquid. To wash the beads, remove the tube from the magnetic stand, add 400 microliters of 0.1-molar sodium hydroxide, 0.05-molar sodium chloride solution, and gently pipette up and down several times.Place the tube back on the magnetic stand, and wait one minute. Then, collect the supernatant. Wash the beads with 200 microliters of 100-millimolar sodium chloride, and remove the supernatant.Add 200 microliters of 20-millimolar tris, resuspend the beads by pipetting, and place the tube on the magnetic stand. After one minute, remove the supernatant. Then, remove the tube from the magnetic stand, add 200 microliters of 1X RNA capture buffer, and resuspend streptavidin magnetic beads by briefly vortexing.Remove 50 microliters of streptavidin magnetic beads, and add it to each labeled RNA tube using a cut pipette tip. Incubate the tubes for 30 minutes at room temperature on a roller. After the tube has been on the magnetic stand for one minute, remove the supernatant.Add 50 microliters of 20-millimolar tris to the beads, and resuspend them. Then, place the tubes into the magnetic stand. After one minute, remove the supernatant.Add 100 microliters of 1X protein RNA binding buffer to the beads, and resuspend them. Then, place the tubes back into the magnetic stand. Once the tubes have been in the stand for one minute, collect the supernatant.Add 100 microliter of master mix B, resuspend by gently pipetting up and down, and avoid creating bubbles. Incubate reaction tubes for 60 minutes at four degrees Celsius on a roller. Move on to the wash and elution steps.Place the tubes into a magnetic stand, collect the flow through, and transfer it into a new tube. Pipette 100 microliters of 1X wash buffer on the beads, resuspend gently, place them back into the stand, wait one minute, and discard the supernatant. Repeat this step two times.Add 40 microliters of elution buffer to the magnetic beads, mix by pipetting up and down, and incubate on a tube shaker. Place the tubes into a magnetic stand, wait one minute, and collect eluate sample. Place on ice, and use for downstream analysis.To demonstrate the purity of the nuclear/cytosolic fractions, proteins were analyzed by Western blot, which showed that alpha-tubulin was only detected in the cytosolic fraction, and PARP protein was detected only in the nuclear fraction. The eluate from the CALB2 three-prime UTR ARE probe demonstrated binding of human R and was absent in the eluate from the mutant probe and the unrelated RNA probe, which binds the iron-responsive protein. To further demonstrate the specificity between the CALB three-prime UTR and human R, the membrane was probed with anti-mesothelin antibody, and all three eluate samples showed no presence of mesothelin, indicating that the stabilizing ARE motif within CALB2 three-prime UTR can specifically bind human R protein.Coomassie staining of the gel shows that equal amounts of proteins were used to incubate with the three different RNA probes. Following this procedure, additional method protein-centric, like immunoprecipitation of a given protein, can be performed in order to address additional question, like which sequence bind to it. After its development, this technique paved the way for researchers in the field of RNA biology to explore possible interacting partners of a specific RNA.

Research

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