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<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>
