Changes
no edit summary
<p> </p>
<p><strong>Obese in Genomics</strong></p>
<p> </p>
<p><strong>What is Obese?</strong></p>
<div>-Obesity is a <a href="https://en.wikipedia.org/wiki/Medical_condition">medical condition</a> in which excess <a href="https://en.wikipedia.org/wiki/Body_fat">body fat</a> has accumulated to the extent that it may have a negative effect on health.</div>
<p> </p>
<p><strong>Arrangement of basic terms in Genomics</strong></p>
<p> </p>
<p><strong>What is Genomics?</strong></p>
</div>
<p> </p>
<p><strong>What is Omics?</strong></p>
<p>General term for a broad discipline of science and engineering</p>
<p>Analyzing the interactions of biological information objects in various <a href="http://omics.org/index.php?title=Omes&action=edit">omes</a> in biology</p>
<p><strong>Main focus</strong></p>
<div>1)mapping information objects such as genes and proteins</div>
<div><strong><u>2)finding interaction relationships among the objects</u></strong></div>
<div>3)engineering the networks and objects to understand and manipulate the regulatory mechanisms</div>
<div> </div>
<div><strong>What is Proteomics?</strong></div>
<div> </div>
<p> </p>
<p>New methods include protein microarrays, <u><strong>immunoaffinity chromatography followed by mass spectrometry(MALDI-TOF mass spectrometry), </strong> </u>and combinations of experimental methods such as phage display and computational methods.</p>
<p> </p>
<p><strong>What is Metabolome?</strong></p>
<p> </p>
<p> </p>
<p><strong>The endogenous metabolome</strong></p>
<p>-> primary metabolome</p>
<p> </p>
<p>[[File:Certificate Jong Bhak 20160427.pdf|700px]]</p> <p> </p> <pstrong>The Human Metabolome Database</strong></p>
<p> </p>
<p><strong><u>Therefore, Focus more on protemoics, Metabolome!</u></strong></p>
<p> -----------------------------------------------------------------------</p>
<p><strong><span style="font-size:14px">Adipose tissue</span></strong></p>
<p> </p>
<p><strong>-> Adipokine </strong></p>
<p> -> <span style="font-size:12px"><span style="color:black; font-family:맑은 고딕">Adipose tissue secreted multiple mediator</span></span></p>
<p><span style="font-size:12px"><span style="color:black; font-family:맑은 고딕"> </span></span>-> Passed through either endocrine or paracrine</p>
<p> Ex: Hormone: leptin, adiponectin</p>
<p> </p>
<p><strong>-> Adiponectin</strong></p>
<p><strong> -> </strong>Adipocyte-secreted adipokine</p>
<p> -> Increase lipid oxidation& anti-inflammatory, insulin-sensitizing, angiogenic action</p>
<p> <strong>=> Anti obesity & Antidiabetic, Decrease insulin resistance </strong></p>
<p> [[File:1.png|400px]]</p>
<p> </p>
<p> </p>
<p> [[File:2.gif|400px]]</p>
<p>-> Illustration of the major physiological and metabolic</p>
<p>processes with which adipose tissue is involved through the secretion</p>
<p>of various adipokines from adipocytes. The interactions may be</p>
<p>autocrine, paracrine, or endocrine.</p>
<p> </p>
<p><span style="font-size:16px"><strong><Searching Scientific Reports></strong></span></p>
<p> [[File:3.png|400px]]</p>
<p> [[File:4.png|400px]]</p>
<p> </p>
<p><strong><span style="font-size:16px"><What is Col6?></span></strong></p>
<p>- COL6 = Collagen type 6</p>
<p>- Abundant constituent of white adipose tissue (WAT)</p>
<p>- COL6 levels positively correlate with hyperglycaemia and insulin resistance</p>
<p>- Composed of three distinct a chains, a1, a2 and a3.(COL6 trimeric building block) and are subsequently secreted into the ECM</p>
<p> </p>
<p><strong><span style="font-size:16px"><What is a3 Chain?></span></strong></p>
<p>-> longest of the three chains</p>
<p>- contains an unusually long N terminus and a globular C5 domain at the C-terminus</p>
<p> </p>
<p>-> C-terminal portion of the a3 subunit is cleaved off during the post-translational processing of COL6 fibrils(COL6a3, Endotrophin)</p>
<p> </p>
<p><strong><span style="font-size:16px"><What is Endotrophin?></span></strong></p>
<p>- Adipokine with potent tumour-promoting effects</p>
<p>- Plays a pivotal role in shaping a metabolically unfavorable microenvironment in adipose tissue during consumption of a high-fat diet (HFD)</p>
<p>- Powerful co-stimulator of pathologically relevant pathways within the ‘unhealthy’ adipose tissue milieu, triggering fibrosis and inflammation and ultimately leading to enhanced insulin resistance& metabolic dysfunction.</p>
<p>- Exerts a major influence in adipose tissue</p>
<p>- Endotrophin within the tumor microenvironment serves as a major mediator of COL6-stimulated mammary tumor growth and subsequent chemo resistance</p>
<p>- Stimulates fibrosis, activates endothelial cell migration and promotes macrophage infiltration into growing solid tumors.</p>
<p>=> elevated mammary tumor expansion and more pronounced metastatic growth</p>
<p> [[File:5.png|400px]]</p>
<p>---------------------------------------------------------</p>
<p><u><strong><span style="font-size:18px">Problem!</span></strong></u></p>
<p> </p>
<p><strong>-> Don’t know the mechanism of how ETP works.</strong></p>
<p> </p>
<p><u><strong><span style="font-size:16px">What I’m going to do!</span></strong></u></p>
<p> </p>
<div><strong>-> Find the Receptor according to the New method of Protemoics.</strong></div>
<div><strong>-> Find the interaction, relationship and mechanisms how they act.(Study of Omics)</strong></div>
<div><u><strong> -> Omics could be applied to genomics perspective!</strong></u></div>
<p>[[File:Self1.png|400px]]</p>
<p> </p>
<p>mETP(204bp, 16.43kda)</p>
<p>ACAGAACCATTGTTTCTCACTAAAACAGATATATGTAAGCTGTCCAGAGATGCTGGGACTT</p>
<p>GTGTGGACTTCAAGTTACTATGGCACTATGACCTAGAGAGCAAAAGTTGCAAGAGATTCTG</p>
<p>GTATGGAGGTTGTGGAGGCAACGAGAACAGATTCCACTCCCAGGAAGAATGTGAAAAGATGTGTAGTCCTGAGTTAACAGTT</p>
<p> </p>
<p>SpyTag(39bp, 16.43kda)</p>
<p>GCCCACATCGTGATGGTGGACGCCTACAAGCCGACGAAG</p>
<p> </p>
<p>pRL(90bp, 7.48kda)</p>
<p>ATGGACAGCAAAGGTTCGTCGCAGAAAGGGTCCCGCCTGCTCCTGCTGCTGGTGGTGTCAAATCTACTCTTGTGCCAGGGTGTGGTCTCC</p>
<p> </p>
<p>(1)</p>
<p> [[File:12.png|600px]]</p>
<p> pRA-GFP-EcoR1-pRL-unknown-mETP-SpyTag-Stop</p>
<p> </p>
<p>-><strong> How to make this cloning?</strong></p>
<p>(1)By Using pRL-EcoR1 forward primer, mETP-SpyTag-Stop-Xho1 primer, make pRL-EcoR1-mETP-SpyTag-Stop-Xho1 by Ex-Tag PCR</p>
<p>[[File:13.png|500px]]</p>
<p>(2) Insert template gained from (1) in T-Vector to check whether it is really pRL-EcoR1-mETP-SpyTag-Stop-Xho1 or not.</p>
<p>(3) Use EcoR1, Xho1 Digestion enzyme to double digest T vector</p>
<p>(4) Double Digest pRA GFP vector(empty vector) and purify it.</p>
<p>(5) ligate (3), (4) product</p>
<p> </p>
<p><strong>-> Detailed on Each Steps</strong></p>
<p>(1) Ex-Tag PCR process</p>
<p> </p>
<p>->Template(pRA-GFP, 20ng): 1ul</p>
<p>->Primer: 1,1ul</p>
<p>->dNTP(10nM): 1ul</p>
<p>->10X Ex-Tag Buffer: 2.5ul</p>
<p>-> Ex-Tag polymerase: 1ul</p>
<p>-> D.W: 17.5ul</p>
<p>----------------------------------------</p>
<p>Total: 25ul</p>
<p> </p>
<p>PCR</p>
<p>->Temperature Gradient : 54,56,58</p>
<p>->98 celsius : 2min</p>
<p>->98 celsius : 10sec</p>
<p>->57 celsius : 30sec</p>
<p>->72 celsius : 30sec(insert 300bp)</p>
<p>->72 celsius : 5min</p>
<p>X35</p>
<p> [[File:14.png|400px]]</p>
<p>-> Can see the insert(300bp) in both 54,56,58 temperature gradient!</p>
<p> </p>
<p> (2)</p>
<p><T vector ligation></p>
<p>Insert DNA mass: 8.607ng(3:1)</p>
<p>2X Rapid ligation: 5ul</p>
<p>T vector: 0.5ul(25ng)</p>
<p>PCR product: 1ul(8.7ng)</p>
<p>D.W: 2.5ul</p>
<p>T4 DNA Ligase: 1ul</p>
<p>----------------------------</p>
<p>Total: 10ul</p>
<p>RT 1 hour incubation</p>
<p>Then, Transformation</p>
<p> </p>
<p><Colony PCR></p>
<p>-> Check whether insert base pairs is inserted in T vector well</p>
<p>T.D.W : 14.9</p>
<p>10X Buffer : 2</p>
<p>M13 primer Forward: 0.5</p>
<p>M13 primer Reverse: 0.5</p>
<p>2.5mM dNTP: 1.6</p>
<p>XL-Taq polymerase: 0.5</p>
<p> ---------------------------------</p>
<p>[[File:15.jpg|400px]]</p>
<p>Can check on 3,5 well(T vector 200bp+ 346bp = 500~600bp)</p>
<p> </p>
<p> (3)</p>
<p>EcoR1 pRL mETP SpyTag Stop Xho1(3,5), pRA_GFP Digestion(EcoR1,Xho1)</p>
<p> [[File:17.png|400px]]</p>
<p>pRA_GFP Digestion(EcoR1,Xho1) EcoR1 pRL mETP SpyTag Stop Xho1(3,5) Extraction</p>
<p> </p>
<p> (4)</p>
<p>EcoR1 pRL mETP SpyTag Stop Xho1 Tvector&pRA_GFP_mETP&pRA_GFP Digestion(EcoR1,Xho1)</p>
<p>[[File:18.png|400px]]</p>
<p> </p>
<p>(5)</p>
<p><Ligation></p>
<p>EcoR1_pRL_mETP_SpyTag_Stop_Xho1 T-Vector</p>
<p>-> insert(9.3ng/ul): 1.5ul</p>
<p>-> vector(19.3ng/ul): 3ul</p>
<p>-> T4 DNA ligase Buffer: 1ul</p>
<p>-> T4 DNA Enzyme: 1ul</p>
<p>-> D.W: 3.5</p>
<p>Transformation</p>
<p> </p>
<p>pRA_GFP EcoR1 pRL mETP SpyTag Stop Xho1 Double Digestion(EcoR1,Xho1)</p>
<p>[[File:19.png|400px]]</p>
<p>------------------------------------------------------------------------------------------</p>
<p>Midi-prep &Transfection</p>
<p> </p>
<p> </p>
<div>1.Opti-MEM+ DNA(each: 3ug)= 250ul</div>
<div>2.(Opti-MEM 241ul+TR 9ul)*2 master mix (vortex well)</div>
<div>3.Put 2 into 1 250ul per each & mix(1min) , stand 30min</div>
<div>4.Treat #3 to H-293 Cell, media change after 10hours</div>
<div>5.Change media to serum free (starvation) Media change after 1day (Check GFP signal)</div>
<div>6.Prepare lysate after 2days(between 24~48hours) (check whether plasmid is well overexpressed by western blot)</div>
<div>7.Put Media 520ul per each in e.tube and 4 celcius 13000rpm, 1min centrifuge, get 500ul of each centrifuged into column and do 45min 13000rpm, 4celcius centrifuge</div>
<p>(Media = conditioned media(CM))</p>
<p>* Store rest of media는 in other tube (labeling)</p>
<p>8. Wash rest of cell in 1X PBS(cold) and after wash, do suction</p>
<p>9. Treat Lysis buffer 200ul and scrape using stripper. Collect all of them in E.tube & labeling, incubation 10 min in cold ice, then 15min 13000rpm, 4celcius centrifuge</p>
<p>(After sup media of centrifuged = cell extract(TCE))</p>
<p> </p>
<p>Transfection</p>
<p> </p>
<p> </p>
<div>1.Opti-MEM+ DNA(each: 3ug)= 250ul</div>
<div>2.(Opti-MEM 241ul+TR 9ul)*2 master mix (vortex well)</div>
<div>3.Put 2 into 1 250ul per each & mix(1min) , stand 30min</div>
<div>4.Treat #3 to H-293 Cell, media change after 10hours</div>
<div>5.Change media to serum free (starvation) Media change after 1day (Check GFP signal)</div>
<div>6.Prepare lysate after 2days(between 24~48hours) (check whether plasmid is well overexpressed by western blot)</div>
<div>7.Put Media 520ul per each in e.tube and 4 celcius 13000rpm, 1min centrifuge, get 500ul of each centrifuged into column and do 45min 13000rpm, 4celcius centrifuge</div>
<p>(Media = conditioned media(CM))</p>
<p>* Store rest of media는 in other tube (labeling)</p>
<p>8. Wash rest of cell in 1X PBS(cold) and after wash, do suction</p>
<p>9. Treat Lysis buffer 200ul and scrape using stripper. Collect all of them in E.tube & labeling, incubation 10 min in cold ice, then 15min 13000rpm, 4celcius centrifuge</p>
<p>(After sup media of centrifuged = cell extract(TCE))</p>
<p> </p>
<p><span style="font-size:14px">Western Blot</span></p>
<p>[[File:Ex1.png|400px]]</p>
<p>tx621(anti-mETP) - (mETP-spytag CM, TCE)</p>
<p> </p>
<p>pRA-mETP-SpyTag Size Exclusion </p>
<p>[[File:Ex2.jpg|400px]]</p>
<p> </p>
<p>(2)pTrc-Spe1-SpyCatcher-Xho1-APEX2</p>
<p>(E.Coli expression vector)</p>
<p> </p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2</p>
<p>(E.Coli expression vector)</p>
<p> </p>
<p>(2) pTrc-Spe1-SpyCatcher-Xho1-APEX2 (E.Coli expression vector)</p>
<p>- APEX2 Sequence(318bp, 26.38kda)</p>
<p>Cgaaagtcttacccaactgtgagtgctgattaccaggacgccgttgagaaggcgaagaagaagctcagaggcttcatcgctgagaagagatgcgctcctctaatgctccgtttggcattccactctgctggaacctttgacaagggcacgaagaccggtggacccttcggaaccatcaagcaccctgccgaactggctcacagcgctaacaacggtcttgacatcgctgttaggcttttggagccactcaaggcggagttccctattttgagctacgccgatttctaccagttggctggcgttgttgccgttgaggtc</p>
<p>- SpyCatcher(385bp, 31.36kda)</p>
<p>ATGGTTGATACCTTATCAGGTTTATCAAGTGAGCA</p>
<p>AGGTCAGTCCGGTGATATGACAATTGAAGAAGATAGTGCTACCCATATTAAATTCTCAAAACGTGATGAG</p>
<p>GACGGCAAAGAGTTAGCTGGTGCAACTATGGAGTTGCGTGATTCATCTGGTAAAACTATTAGTACATGGA</p>
<p>TTTCAGATGGACAAGTGAAAGATTTCTACCTGTATCCAGGAAAATATACATTTGTCGAAACCGCAGCACC</p>
<p>AGACGGTTATGAGGTAGCAACTGCTATTACCTTTACAGTTAATGAGCAAGGTCAGGTTACTGTAAATGGC</p>
<p>AAAGCAACTAAAGGTGACGCTCATATTTAAATGGTTGATGCTTGAGGATCCGAATTCGAGCTCCGTCGAC</p>
<p>[[File:Ex3.jpg|400px]]</p>
<p> </p>
<p>(2) pTrc-Spe1-SpyCatcher-Xho1-APEX2 (E.Coli expression vector) sequencing data</p>
<p>[[File:Ex4.png|400px]]</p>
<p>*pTRC sequencing primer</p>
<p>F: 5’-AGCTGTTGACAATTAATCATCCGGC-3’</p>
<p>R: 5'-TCTGCGTTCTGATTTAATCTGTATCAGGC-3‘</p>
<p> </p>
<p>(2)pTRC_APEX2_SpyCatcherSpe1Xho1(Spe1-SpyCatcher-Xho1)</p>
<p>[[File:Ex5.png|400px]]</p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2(39.1kda) expected sequence</p>
<p>[[File:Ex6.png|400px]]</p>
<p> </p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2 (E.Coli expression vector)</p>
<p>1.Add Spe1, Xho1 between mETP</p>
<p> </p>
<p>PCR(Ex-Nrg1)</p>
<p>-TDW :37.75ul</p>
<p>-10X EX Taq Buffer : 5ul</p>
<p>-Primer(R/F): 1ul(per each)</p>
<p>-Template(mETP): 1ul(10ng) -----------X4(temperature gradient: 54,56,58,60)</p>
<p>-2.5mM dNTP MIX: 4ul</p>
<p>-Ex Taq Polymerase: 0.25ul</p>
<p>------------------------------</p>
<p> 50ul</p>
<p> </p>
<p>Purify</p>
<p> </p>
<p>-Use mini-prep kit</p>
<p> (PW buffer 650ul, product 50ul in column, 30sec centrifuge, discard bottom one and 1min centrifuge, transfer column to1.5ml tube, 2nd D.W 35ml , stand 1min, 1min centrifuge</p>
<p>->purified mETP Ex-Tag PCR product(56,58,60)</p>
<p>[[File:Ex7.jpg|400px]]</p>
<p>mETP(Spe1-mETP-Xho1) ExTaq gradient PCR(54,.56,58,60)</p>
<p> </p>
<p><T vector Cloning></p>
<p>-> mETP Ex-Taq PCR (each end A exists) ligate into T vector(=pGEM-T easy vector, each end T exists), Transformation to E.coli</p>
<p> </p>
<p>-Ligation</p>
<p>-</p>
<p>->NEB Calculator site(nebiocalculator.net.com/#!/ligation) insert length(mETP =204, primer, total 222bp), vector length(T vector :3015bp), T vector mass</p>
<p>->3:1=> insert 5.522ng</p>
<p> </p>
<p>-> 2X Rapid Ligation buffer: 5ul</p>
<p> T vector: 0.5ul</p>
<p> PCR product(Insert):</p>
<p> D.W :</p>
<p>T4 DNA Ligase: 1ul</p>
<p>---------------------------------------</p>
<p> Total: 10ul</p>
<p>1hour RT incubation</p>
<p> </p>
<p>[[File:Ex8.jpg|400px]]</p>
<p><Colony PCR></p>
<p>T.D.W : 14.9</p>
<p>10X Buffer : 2</p>
<p>M13 primer Forward: 0.5</p>
<p>M13 primer Reverse: 0.5</p>
<p>2.5mM dNTP: 1.6</p>
<p>XL-Taq polymerase: 0.5</p>
<p> </p>
<p>Phusion Colony PCR</p>
<p> </p>
<p>Replica 4,7</p>
<p>Double Digestion</p>
<p>(1) pTrc Vector(pTrc-Spycatcher-APEX2,3410ng/ul): 5ug(1.5ul)</p>
<p>Enzyme(Spe1, Xho1): each 1.5ul</p>
<p>Buffer(Cutsmart,10X):1ul</p>
<p>T.D.W:4.5ul</p>
<p>------------------------------------------------------</p>
<p>10ul</p>
<p> </p>
<p>(2) pTrc Vector( expected to be Spycatcher Cut ,15ng/ul): 15ul(225ng)</p>
<p>Enzyme(Spe1, Xho1): 1ul(each)</p>
<p>Buffer(Cutsmart,10X): 2ul</p>
<p>T.D.W:1</p>
<p>------------------------------------------------------</p>
<p>20ul</p>
<p> </p>
<p>(3) mETP T-vector</p>
<p>-> Restriction Enzyme(Spe1, Xho1): 1.5ul 씩</p>
<p>-> Tvector(735ng/ul): 6.8ul(5ng)</p>
<p>-> 10X Buffer: 2ul</p>
<p>-> T.D.W: 8.2ul</p>
<p>--------------------------------------------------</p>
<p> 20ul</p>
<p> </p>
<p>37 celcius incubation</p>
<p>[[File:Ex9.png|400px]]</p>
<p> </p>
<p>Ligation</p>
<p> </p>
<p> </p>
<p>-> Insert: 212bp</p>
<p>-> Vector DNA mass: 50ng</p>
<p>-> 3:1</p>
<p>=> Insert DNA mass: 7.227ng</p>
<p> </p>
<p>10X T4 DNA Ligase buffer: 3ul</p>
<p>Vector DNA: 50ng(17ul)</p>
<p>Insert DNA: 7.227ng(2.06ul)</p>
<p>D.W: 6.94ul</p>
<p>T4 DNA Ligase: 1ul</p>
<p>---------------------------------------------------------------</p>
<p>Total: 30ul</p>
<p>RT incubation</p>
<p> </p>
<p>->Transformation</p>
<p> </p>
<p>-> Colony: 4</p>
<p>-> incubation</p>
<p>->Mini-prep</p>
<p>-> 1.5% gel, 4 Digestion</p>
<p>[[File:Ex10.png|400px]]</p>
<p>pTrc Spe1-mETP-Xho1 colony(1,2,3,4)</p>
<p>Double Digestion</p>
<p> </p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2 (E.Coli expression vector) sequencing result</p>
<p>[[File:Ex11.png|400px]]</p>
<p> </p>
<p>(2) pTrc-Spe1-SpyCatcher-Xho1-APEX2 (E.Coli expression vector)</p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2 (E.Coli expression vector) sequencing result</p>
<p> </p>
<p> </p>
<p>(1)Protein expression by transformation on Ecoli(BL21)</p>
<p>*Store each of E.coli in -80 celcius Deep Freezer</p>
<p> </p>
<p>(2)Do IPTG induction test(small scale) to check whether it is soluble, overexpress or not</p>
<p> </p>
<p>(3) Get protein & purify!</p>
<p> </p>
<p><Protein induction & soluble test protocol></p>
<p> </p>
<p>1.Incubate E.coli with 5ml+antibiotic LB (previous day)</p>
<p>●</p>
<p>2.Dilute up to 1:50(2%) and incubate with new bottle</p>
<p>●</p>
<p>3.Incuabte until OD(optical density) reach 0.5~0.8 at 600nm. Then, divide it into 1.5ml tube 200ul tube(IPTG(-), store at 4 celcius)</p>
<p>●</p>
<p>4.Treat IPTG(=molecular biology reagent) (final: 0.5mM/L , 1M*xL = 0.5mM*yL)</p>
<p>Ex: For 200ml, 100ul 1M IPTG is needed</p>
<p> </p>
<p>5. Incubate ITPG treated with 37 celcius shaker about 3.5 hours</p>
<p> </p>
<p>6. Divide it into 1.5ml tube 200ul (14000rpm,1min centrifuge, label IPTG(+) Sup to Sup(supernatant, Wash the ppt(pallet) with T.D.W 50ul and label it to IPTG(+) PPT and store both with 4 celcius )</p>
<p> </p>
<p>7. Centrifuge rest of them (4000rpm, 15min) & remove sup</p>
<p> </p>
<p>8. Resuspension it with Phosphate buffer(=Ni-NTA wash buffer, (20mM Tris HCl(pH 8), 150mM NaCl, 20mM Imidazole))</p>
<p> </p>
<p>9. centrifuge, remove sup</p>
<p>10. Resuspension each with (1L: 30~35ml wash buffer )</p>
<p> </p>
<p>11. Treat Lysozyme(50ul per 1L) and incubate it with shaking (0.5~1h RT)</p>
<p> </p>
<p>12. sonication(20 amplitude, 10min(processing time : 5min)</p>
<p> </p>
<p>13. Take 50ul and divide it into ppt&SUP to check solubility</p>
<p> </p>
<p>14. Centrifuge Rest of them except 50ul with 10000rpm, isolate Sup and ppt. Label each of them with IPTG(+) sonication(+) sup, ppt, IPTG(+) sonication(-) sup, ppt</p>
<p> </p>
<p>15. Mix Labeled with 5X SDS buffer and boil it with 100 celcius(5~10min)</p>
<p> </p>
<p>* Store it in -20 celcius</p>
<p> </p>
<p>pTrc-Spe1-mETP-Xho1-APEX2 protein size</p>
<p>Open reading frame(ORF): 1068bp, 355 amino acids, 39.1kDa</p>
<p> </p>
<p>Þ10% gel commassie blue(60v->120v)</p>
<p>[[File:Ex12.png|400px]]</p>
<p> </p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2 protein his column purification</p>
<p>(E.Coli expression vector)</p>
<p>[[File:Ex13.jpg|400px]]</p>
<p> </p>
<p>His column purification Comassie blue staining</p>
<p>[[File:Ex14.png|400px]]</p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2 protein</p>
<p> </p>
<p>#19 size exclusion(purification)</p>
<p>[[File:Ex15.jpg|400px]]</p>
<p>(3) pTrc-Spe1-mETP-Xho1-APEX2 protein</p>
<p> </p>
<p>(1)pRA-GFP-pRL-mETP-SpyTag-Stop-Xho1 (mammalian expression vector)</p>
<p>& (2) pTrc-Spe1-SpyCatcher-Xho1-APEX2</p>
<p>(E.Coli expression vector) </p>
<p>Binding test</p>
<p>(protein to protein)</p>
<p>[[File:Ex16.png|400px]] [[File:Ex17.jpg|400px]] </p>
<p>* pRA-mETP-SpyTag_CM 10ul(concentrated)</p>
<p>pTrc-Spycatcher 200ng, 500ng, 100ng, 200ng, 500ng, 1000ng</p>
<p> binding test(Anti His) (15%)</p>
<p>* pRA-mETP-SpyTag_CM 10ul(concentrated)</p>
<p>pTrc-Spycatcher 200ng, 500ng, 100ng, 200ng, 500ng, 1000ng</p>
<p>binding test(Anti mETP(Tx621))(15%)</p>
<p> </p>
<p>Further study & Prospect</p>
<p> </p>
<p>-Do protein interaction experiment in cell to see whether it is well connected in vitro</p>
<p>-</p>
<p>-</p>
<p>-Find the protein that is correlated with mETP through ‘mass spectroscopy’ technique</p>
<p> </p>
<p> -> Therefore, we can identify unknown ‘protein’ and its gene and their location.</p>
<p> </p>
<p>-Applied to Genomics</p>
<p> </p>
<p> -> Analyze how different, how overexpressed, how different of unknown ‘protein’ gene between obese people to lean people by genome sequencing of certain region of unknown ‘protein’ gene</p>
<p> </p>
<p> </p>
<p>Reference</p>
<p> </p>
<p>- Definition of Proteomics <a href="http://biolecture.org/index.php/Proteomics">http://</a><a href="http://biolecture.org/index.php/Proteomics">biolecture.org/index.php/Proteomics</a></p>
<p>- Definition of Omics <a href="http://biolecture.org/index.php/Omics">http</a><a href="http://biolecture.org/index.php/Omics">://</a><a href="http://biolecture.org/index.php/Omics">biolecture.org/index.php/Omics</a></p>
<p>-Bhak, J. (2016, 6 13). Openfree biolecture. Retrieved from Openfree biolecture: <u><a href="http://biolecture.org/index.php/SELF:_Self_evaluating_learning_framework">http://biolecture.org/index.php/SELF:_Self_evaluating_learning_framework</a></u></p>
<p>-Endotrophin triggers adipose tissue fibrosis and metabolic dysfunction. <u><a href="https://www.ncbi.nlm.nih.gov/pubmed/24647224">https://www.ncbi.nlm.nih.gov/pubmed/24647224</a></u></p>
<p>-Adipocyte-derived endotrophin promotes malignant tumor progression</p>
<p><u><a href="https://www.jci.org/articles/view/63930">https://www.jci.org/articles/view/63930</a></u></p>
<p>-Secrets of a covalent interaction for biomaterials and biotechnology: SpyTag and SpyCatcher.</p>
<p><u><a href="https://www.ncbi.nlm.nih.gov/pubmed/26517567">https://www.ncbi.nlm.nih.gov/pubmed/26517567</a></u></p>
<p>-Directed evolution of APEX2 for electron microscopy and proximity labeling.</p>
<p><u><a href="https://www.ncbi.nlm.nih.gov/pubmed/25419960">https://www.ncbi.nlm.nih.gov/pubmed/25419960</a></u></p>
<p>-From genomics to proteomics (Nature Review)</p>
<p><u><a href="http://www.nature.com/nature/journal/v422/n6928/full/nature01510.html">http://www.nature.com/nature/journal/v422/n6928/full/nature01510.html</a></u></p>
<p>-Innovation: Metabolomics: the apogee of the omics trilogy</p>
<p><u><a href="http://www.nature.com/nrm/journal/v13/n4/abs/nrm3314.html">http://www.nature.com/nrm/journal/v13/n4/abs/nrm3314.html</a></u></p>
<p>-Mass-spectrometric exploration of proteome structure and function</p>
<p><u><a href="http://www.nature.com/nature/journal/v537/n7620/abs/nature19949.html">http://www.nature.com/nature/journal/v537/n7620/abs/nature19949.html</a></u></p>
<p> </p>