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Synthesis of PHP-TREN-B1-Peptide
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== Peptides<br> == {| border="1" width="711" cellspacing="1" cellpadding="1" |- | width="200" | Name<br> | Seq<br> | Form<br> | Transfection Efficiency<br> |- | width="200" | Tat<sup>48–60</sup> | GRKKRRQRRRPPQ | <br> | <br> |- | width="200" | Tat<sup>49-60</sup><br> | RKKRRQRRRPPQ<br> | <br> | <br> |- | width="200" | Tat<sup>47–57</sup><br> | RKKRRQRRR-GGG<br> | <br> | <br> |- | width="200" | Tat2<br> | C(YGRKKRRQRRRG)2<br> | <br> | <br> |- | width="200" | HIV Rev<br> | TRQARRNRRRRWRERQR<br> | <br> | <br> |- | width="200" | Penetratin (Antp 43–58 ) | RQIKIWFQNRRMKWKK | <br> | <br> |- | width="200" | Transportan | GWTLNSAGYLLGKINLKALAALAKKIL | <br> | <br> |- | width="200" | TP10 | AGYLLGKINLKALAALAKKIL | <br> | <br> |- | width="200" | Oligoarginine (R8) | RRRRRRRR | <br> | <br> |- | width="200" | MAP | KLALKLALKALKAALKLA | <br> | <br> |- | width="200" | MPG | GALFLGFLGAAGSTMGAWSQPKKKRKV | <br> | <br> |- | width="200" | MPGa | GALFLAFLAAALSLMGLWSQPKKKRKV | <br> | <br> |- | width="200" | CADY<br> | GLWRALWRLLRSLWRLLWRA-cya<br> | <br> | <br> |- | width="200" | LK15<br> | RKKRRQRRRGGGKLLKLLLKLLLKLLK-CONH<sub>2</sub><br> | <br> | <br> |- | width="200" | KALA <br> | WEAKLAKALAKALAKHLAKALAKALKACEA<br> | <br> | <br> |- | width="200" | Pep1 (Chariot)<br> | KETWWETWWTEWSQPKKKRKV<br> | <br> | <br> |- | width="200" | INF-7<br> | GLFEAIEGFIENGWEGMIDGWYG<br> | <br> | <br> |- | width="200" | GALA-INF3 | GLFEAIEGFIENGWEGLAEALAEALEALAA | <br> | <br> |- | width="200" | HA2 | GLFGAIAGFIENGWEGMIDGWYG | <br> | <br> |- | width="200" | Tat<br> | RKKRRQRRRPPQ<br> | <br> | 4X10<sup>6</sup><br> |- | width="200" | Tat-CTHD<br> | RKKRRQRRRPPQC<br> | C-Terminal disulfide homodimer<br> | 1.2X10<sup>7</sup><br> |- | width="200" | Tat-NTHD<br> | CRKKRRQRRRPPQ<br> | N-Terminal disulfide homodimer<br> | 1.0X10<sup>7</sup><br> |- | width="200" | Cyclic-Tat<br> | CRKKRRQRRRPPQC<br> | Disulfide bond between C-terminal and N-terminal<br> | 3.8X10<sup>7</sup><br> |} <br> == Synthesis PHP-TREN-B1-Peptide == '''SPDP''': Succinimidyl 3-(2-pyridyl-dithio) propionate, NHS-Pyridyldithiol Crosslinkers, Water-insoluble (dissolve first in DMF or DMSO), SPDP crosslinker is membrane permeable, Pierce $133/50mg<br> [[Image:PHP-TREN-B1 SPDP Peptide.png]]<br> <br> 6.6 mg of PEI (2kD) in 0.5 mL phosphate buffer (pH 8.0) <br> Add 0.5 mL of DMSO solution containing 3.8 mg of SPDP<br> Stir overnight at room temperature.<br> Dialyse the reaction mixture against water at 4 C overnight using a Spectra/Por dialysis membrane (MWCO 1000).<br> Freeze-dry the solution--> 2.2 mg of PEI-SPDP<br> Add 2.0mg peptide to 0.9 mg PEI-SPDP in 1.0 mL of PBS. <br> React overnight at room temperature.<br> Dialyse the reaction mixture overnight using a Spectra/Por membrane (MWCO 1000).<br> Freeze-dry yielding a product.<br> Before use, dissolve PEI-Peptide conjugatein pure water.<br> <br> '''SMCC:''' NHS-Maleimide Crosslinkers, Non-cleavable Water-insoluble (dissolve first in DMF or DMSO), $90/50 mg<br> [[Image:Smcc conjugation.jpeg]]<br> '''Sulfo-SMCC'''<br> [[Image:Sulfo-SMCC-conjugation.jpg]]<br> <br> === Synthesis of PHP-TREN-B1<br> === '''10/25/14, Synthesis'''<br> 1.5 g DAP + 3.6 g DBP + 1.5 ml MeOH, dissolve, feel heat<br> +(200 ul TREN + 0.5 ml MeOH)<br> mix, 50 oC react o/n<br> add 1 ml MeOH, 60 oC react 3 hour<br> '''10/26/14 Purification''': + H2O to 10 ml, + 40 ml isopropanol, spin, repeat once.<br> Dissolve in H2O to 10 ml<br> '''10/30/14, Modification'''<br> Make Sulfo-SMCC: dissolve 5 mg Sulfo-SMCC in 50 ul DMSO. Final conc: 0.1 mg/ul<br> {| border="1" width="718" cellspacing="1" cellpadding="1" |- | Code<br> | Name<br> | Modification<br> | Peptide<br> | Peptide Sequence<br> |- | P<br> | PHP-TREN-B1<br> | 20 ul P<br> | <br> | <br> |- | S<br> | PHP-TREN-B1-Cho <br> | 20 ul P+ 5 ul 0.5 ug/ul Cho-Chl<br> | <br> | <br> |- | T<br> | PHP-TREN-B1-Gu | 20 ul P + ~5 mg pyrozole<br> | <br> | <br> |- | U1<br> | PHP-TREN-B-5H-Tat-5H-C | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''5.7''' mg/ul peptide<br> | 5H-Tat-5H-C<br> | HHHHHGRKKRRQRRRPPAGHHHHHC<br> |- | U2<br> | PHP-TREN-B1-HA2-R | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.2''' mg/ul peptide <br> | HA2-R9<br> | GDIMGEWGNEIFGAIAGFLGGGRRRRRRRRRGC<br> |- | U3<br> | PHP-TREN-B1-Tat-IFN7 | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.1''' mg/ul peptide | Tat-IFN7<br> | RRRQRRKKRGLFEAIEGFIGNGWEGMIDGWYGC<br> |- | U4<br> | PHP-TREN-B1-R9-HA | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.4''' mg/ul peptide | R9-HA<br> | RRRRRRRRRGLFEAIEGFIENGWEGMIDGWYGC<br> |- | U5<br> | PHP-TREN-B1-Tat-HA2 | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.3''' mg/ul peptide <br> | Tat-HA2<br> | RRRQRRKKRGGDIMGEWGNEIFGAIAGFLGC, $215/mg<br> |- | H6<br> | PHP-TREN-B1-C-5H-Tat-5H-C<br> | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of 5.6 mg/ul peptide<br> | C-5H-Tat-5H-C<br> | CHHHHHGRKKRRQRRRPPAGHHHHHC<br> |- | U7<br> | PHP-TREN-B1-Tat2<br> | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add '''2.5''' ul of '''10.5''' mg/ul peptide<br> | Tat2<br> | C(YGRKKRRQRRRG)2<br> |} <br> '''10/30/14: Transfection'''<br> 293T, 24 wells, 1 ug DNA, 3 ul diluted TR: 1;30, 1:50, 1:75<br> '''10/31/14 Result'''<br> [[Image:PHP-TREN-B1 peptide 10 31 2014.png]]<br> 11/1/14 [[Image:PHP-TREN-B1 peptide 11 1 2014.JPG]] [[Image:PHP-TREN-B1 peptide 11 1 2014 2.JPG]] 11/2/14<br> [[Image:PHP-TREN-B1 peptide 11 2 2014.JPG]]<br> <br> 11/3/14 [[Image:PHP-TREN-B1 peptide 11 3 2014 2.JPG]] '''<br>''' '''2nd Transfection'''<br> 11/2/14 [[Image:PHP-TREN-B1 peptide 11 2 2014 2.JPG]]<br> 11/3/14 contaimination--> no or few green cells [[Image:PHP-TREN-B1 peptide 11 3 2014.JPG]] <br> === Synthesis of PHP-TREN-B1-Cho-Gu-Peptide<br> === '''Synthesis'''<br> 10/9/14 1.5g DAP, 3.6 g DBP + 1.5 ml MeOH, dissolve 196 mg TREN (200 ul) + 1.5 ml MeOH react o/n at 50 oC purify w/ 5X 5 ml MeOH dissolve in 12 ml H2O, store at 4 oC--> turbid + 600 ul water soluble PEI18-Cho + 80 mg DSP mix, react o/n +0.5 g Pyrozale Make Sulfo-SMCC: dissolve 5 mg Sulfo-SMCC in 50 ul DMSO. Final conc: 0.1 mg/ul<br> {| border="1" width="718" cellspacing="1" cellpadding="1" |- | Code<br> | Name<br> | Modification<br> | Peptide<br> | Peptide Sequence<br> |- | Q<br> | PHP-TREN-B1-Cho-Gu<br> | 20 ul Q PHP-TREN-B1-Cho-Gu, + 10 ul H2O<br> | <br> | <br> |- | V1<br> | PHP-TREN-B1-Cho-Gu-5H-Tat-5H-C | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''5.7''' mg/ul peptide<br> | 5H-Tat-5H-C<br> | HHHHHGRKKRRQRRRPPAGHHHHHC<br> |- | V2<br> | PHP-TREN-B1-Cho-Gu-HA2-R | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.2''' mg/ul peptide <br> | HA2-R9<br> | GDIMGEWGNEIFGAIAGFLGGGRRRRRRRRRGC<br> |- | V3<br> | PHP-TREN-B1-Cho-Gu-Tat-IFN7 | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.1''' mg/ul peptide | Tat-IFN7<br> | RRRQRRKKRGLFEAIEGFIGNGWEGMIDGWYGC<br> |- | V4<br> | PHP-TREN-B1-Cho-Gu-R9-HA | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.4''' mg/ul peptide | R9-HA<br> | RRRRRRRRRGLFEAIEGFIENGWEGMIDGWYGC<br> |- | V5<br> | PHP-TREN-B1-Cho-Gu-Tat-HA2 | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of '''2.3''' mg/ul peptide <br> | Tat-HA2<br> | RRRQRRKKRGGDIMGEWGNEIFGAIAGFLGC, $215/mg<br> |- | V6<br> | PHP-TREN-B1-Cho-Gu-C-5H-Tat-5H-C<br> | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of 5.6 mg/ul peptide<br> | C-5H-Tat-5H-C<br> | CHHHHHGRKKRRQRRRPPAGHHHHHC<br> |- | V7<br> | PHP-TREN-B1-Cho-Gu-Tat2<br> | 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add '''2.5''' ul of '''10.5''' mg/ul peptide<br> | Tat2<br> | C(YGRKKRRQRRRG)2<br> |} <br> <br> '''Transfection''' 11/2/14 [[Image:PHP-TREN-B1 peptide 11 2 2014 3.JPG]] contamination-->no blue cells 11/3/14 [[Image:PHP-TREN-B1 peptide 11 3 2014 3.JPG]] <br> 11/7/14 Transfection: ~60% cell density resulted a lot of cell death because lower cell density take pictures on 11/13/14 {| border="1" width="200" cellspacing="1" cellpadding="1" |- | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 |- | P | P | P | U1 | U1 | U1 |- | U2 | U2 | U2 | U3 | U3 | U3 |- | U4 | U4 | U4 | U5 | U5 | U5 |- | U6 | U6 | U6 | U7 | U7 | U7 |} 11/13/14<br> [[Image:P U 2014 11 12.JPG]] 11/14/14 [[Image:P U 2014 11 14.JPG]] 11/15/14 [[Image:P U 2014 11 15 1.JPG]] {| border="1" width="200" cellspacing="1" cellpadding="1" |- | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 |- | Q | Q | Q | V1 | V1 | V1 |- | V2 | V2 | V2 | V3 | V3 | V3 |- | V4 | V4 | V4 | V5 | V5 | V5 |- | V6 | V6 | V6 | V7 | V7 | V7 |} 11/13/14<br> [[Image:Q V 2014 11 12.JPG]] <br> 11/14/14 [[Image:Q V 2014 11 14.JPG]] 11/15/14 [[Image:Q V 2014 11 15.JPG]] {| border="1" width="200" cellspacing="1" cellpadding="1" |- | 1:30 | 1:50 | 1:75 | 1:30 | 1:50 | 1:75 |- | S | S | S | T | T | T |} 11/13/14<br> [[Image:S T 2014 11 12.JPG]] 11/14/14 [[Image:S T 2014 11 14.JPG]] 11/15/14 [[Image:S T 2014 11 15.JPG]] Transfection again on 7/12/14 with higher cell density picture on 7/13/14 {| border="1" width="200" cellspacing="1" cellpadding="1" |- | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 |- | P | P | P | U1 | U1 | U1 |- | U2 | U2 | U2 | U3 | U3 | U3 |- | U4 | U4 | U4 | U5 | U5 | U5 |- | U6 | U6 | U6 | U7 | U7 | U7 |} 11/13/14<br> [[Image:P U 2014 11 13.JPG]] 11/14/14 [[Image:P U 2014 11 14 1.JPG]] 11/15/14 [[Image:P U 2014 11 15.JPG]] 11/16/14 [[Image:P U 2014 11 16.JPG]] {| border="1" width="200" cellspacing="1" cellpadding="1" |- | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:75 |- | Q | Q | Q | V1 | V1 | V1 |- | V2 | V2 | V2 | V3 | V3 | V3 |- | V4 | V4 | V4 | V5 | V5 | V5 |- | V6 | V6 | V6 | V7 | V7 | V7 |} 11/13/14<br> [[Image:Q V 2014 11 13.JPG]] <br> <br> 11/14/14<br> [[Image:Q V 2014 11 14 1.JPG]] 11/15/14<br> [[Image:Q V 2014 11 15 1.JPG]] 11/16/14 [[Image:Q V 2014 11 16.JPG]] === Synthesis of PHP-TREN-B1-PEI18-Cho: W === '''Synthesis''' 10/16/14 1.5g DAP, 3.6 g DBP + 1.5 ml MeOH, dissolve 196 mg TREN (200 ul) + 1.5 ml MeOH react o/n at 50 oC purify w/ 20 ml EtOH, 2X<br> dissolve in 12 ml H2O <br> + 305 ul water soluble PEI18-Cho (concentrated 10/5/14, original vol=27.5 ml, concentrated vol=17.5 ml), + 80 mg DSP, react overnight at RT.<br> Dialyse vs water, freeze dry, dissolve dried pellet in 10 ml water. --> '''W '''<br> {| border="1" width="200" cellspacing="1" cellpadding="1" |- | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 | bgcolor="#ffffcc" | 1:30 | bgcolor="#ffffcc" | 1:50 |- | S | S | T | T | W | W |} 11/13/14<br> [[Image:S T W 2014 11 13.JPG]]<br> 11/14/14 [[Image:S T W 2014 11 14.JPG]] 11/15/14 [[Image:S T W 2014 11 15.JPG]] 11/16/14 [[Image:S T W 2014 11 16.JPG]] <br> '''New Transfection'''. 2014/11/19 P, U1 to U7 2014/11/20 [[Image:P U 2014 11 20.JPG]] 2014/11/21 [[Image:P U 2014 11 21.JPG]] 2014/11/22 [[Image:P U 2014 11 22.JPG]] <br> Q, V1 to V7 2014/11/20 [[Image:Q V 2014 11 20.JPG]] 2014/11/21 [[Image:Q V 2014 11 21.JPG]] 2014/11/22 [[Image:Q V 2014 11 22.JPG]] S,T, W, Turbofect, L2K, B1 2014/11/20 [[Image:S T W 2014 11 20.JPG]] 2014/11/21 [[Image:S T W 2014 11 21.JPG]] 2014/11/22 [[Image:S T W 2014 11 22.JPG]] <br> == Transfection using Several Cell Lines == === 293E === Date: 11/27/14, P, U1~U7, 1:30, 1:50, 1:75 [[Image:293E P U 11 27 14.JPG]] 11/27/14, Q, V1~V7, 1:30, 1:50, 1:75 [[Image:293E Q V 11 27 14.JPG]] 11/27/14, W, P, Q, R, X, L2K [[Image:293E W P Q R X 11 27 14.JPG]] === Hela === Transfection Date: 11/25/14 Picture of 11/27/14 Q, V1 ~V7, 1:30, 1:50, 1:75 [[Image:Hela Q V 11 27 14.JPG]] 11/30/14 [[Image:Hela Q V 11 30 14.JPG]] 11/27/14, P, U1~U7, 1:30, 1:50, 1:75 [[Image:Hela P U 11 27 14.JPG]] 11/30/14 [[Image:P U 2014 11 30.JPG]] 11/27/14, W (1:30, 1:50, 1:75), L2K, X, P, Q (1:30, 1:50), R (1:30, 1:50), B1 [[Image:Hela W P Q R X 11 27 14.JPG]] 11/30/14 [[Image:Hela W P Q R X 11 30 14.JPG]] <br> == Transfection of CHO cells == Transfection reagents: P, Q, R, W, 1 ug DNA {| border="1" width="499" cellspacing="1" cellpadding="1" |- | '''P''' 1:30, 3 ul | 1:50, 3 ul | 1:75, 3 ul | '''Q''' 1:30, 3 ul | 1:50, 3 ul | 1:75, 3 ul |- | 1:30, 6 ul | 1:50, 6 ul | 1:75, 6 ul | 1:30, 6ul | 1:50, 6 ul | 1:75, 6 ul |- | '''R''' 1:30, 3 ul | 1:50, 3 ul | 1:75, 3 ul | '''W''' 1:30, 3ul | 1:50, 3ul | 1:75, 3ul |- | 1:30, 6 ul | 1:50, 6 ul | 1:75, 6 ul | 1:30, 6ul | 1:50, 6ul | 1:75, 6 ul |} <br> Replicates: 2 plates 12/12/2014: 3 days after transfection plate 2 [[Image:12 12 14 1.JPG]] plate 1, [[Image:12 12 14 2.JPG]] <br> [[Image:12 12 14 3 s.JPG]] <br> [[Image:12 12 14 4 s.JPG]] 2014/12/15 [[Image:12_14_14_1.JPG]] [[Image:12_14_14_2.JPG]] [[Category:Paul]] [[Category:Transfection]]
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