Paul.xu at 23:21, 15 December 2014

2014-12-15T23:21:21Z

New page

== Peptides ==

{| border="1" width="711" cellspacing="1" cellpadding="1"
|-
| width="200" | Name 
| Seq 
| Form 
| Transfection Efficiency 
|-
| width="200" | Tat48–60
| GRKKRRQRRRPPQ
| 
| 
|-
| width="200" | Tat49-60 
| RKKRRQRRRPPQ 
| 
| 
|-
| width="200" | Tat47–57 
| RKKRRQRRR-GGG 
| 
| 
|-
| width="200" | Tat2 
| C(YGRKKRRQRRRG)2 
| 
| 
|-
| width="200" | HIV Rev 
| TRQARRNRRRRWRERQR 
| 
| 
|-
| width="200" | Penetratin (Antp 43–58 )
| RQIKIWFQNRRMKWKK
| 
| 
|-
| width="200" | Transportan
| GWTLNSAGYLLGKINLKALAALAKKIL
| 
| 
|-
| width="200" | TP10
| AGYLLGKINLKALAALAKKIL
| 
| 
|-
| width="200" | Oligoarginine (R8)
| RRRRRRRR
| 
| 
|-
| width="200" | MAP
| KLALKLALKALKAALKLA
| 
| 
|-
| width="200" | MPG
| GALFLGFLGAAGSTMGAWSQPKKKRKV
| 
| 
|-
| width="200" | MPGa
| GALFLAFLAAALSLMGLWSQPKKKRKV
| 
| 
|-
| width="200" | CADY 
| GLWRALWRLLRSLWRLLWRA-cya 
| 
| 
|-
| width="200" | LK15 
| RKKRRQRRRGGGKLLKLLLKLLLKLLK-CONH2 
| 
| 
|-
| width="200" | KALA 
| WEAKLAKALAKALAKHLAKALAKALKACEA 
| 
| 
|-
| width="200" | Pep1 (Chariot) 
| KETWWETWWTEWSQPKKKRKV 
| 
| 
|-
| width="200" | INF-7 
| GLFEAIEGFIENGWEGMIDGWYG 
| 
| 
|-
| width="200" | GALA-INF3
| GLFEAIEGFIENGWEGLAEALAEALEALAA
| 
| 
|-
| width="200" | HA2
| GLFGAIAGFIENGWEGMIDGWYG
| 
| 
|-
| width="200" | Tat 
| RKKRRQRRRPPQ 
| 
| 4X106 
|-
| width="200" | Tat-CTHD 
| RKKRRQRRRPPQC 
| C-Terminal disulfide homodimer 
| 1.2X107 
|-
| width="200" | Tat-NTHD 
| CRKKRRQRRRPPQ 
| N-Terminal disulfide homodimer 
| 1.0X107 
|-
| width="200" | Cyclic-Tat 
| CRKKRRQRRRPPQC 
| Disulfide bond between C-terminal and N-terminal 
| 3.8X107 
|}

 

== 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 

[[Image:PHP-TREN-B1 SPDP Peptide.png]] 

 

6.6 mg of PEI (2kD) in 0.5 mL phosphate buffer (pH 8.0)  

Add  0.5 mL of DMSO solution containing 3.8 mg of SPDP 

Stir overnight at room temperature. 

Dialyse the reaction mixture against water at 4 C overnight using a Spectra/Por dialysis membrane (MWCO 1000). 

Freeze-dry the solution--> 2.2 mg of PEI-SPDP 

Add 2.0mg peptide to  0.9 mg PEI-SPDP  in 1.0 mL of PBS. 

React overnight at room temperature. 

Dialyse the reaction mixture overnight using a Spectra/Por membrane (MWCO 1000). 

Freeze-dry yielding a product. 

Before use, dissolve PEI-Peptide conjugatein pure water. 

 

'''SMCC:''' NHS-Maleimide Crosslinkers, Non-cleavable Water-insoluble (dissolve first in DMF or DMSO), $90/50 mg [[Image:Smcc conjugation.jpeg]] 

'''Sulfo-SMCC''' 

[[Image:Sulfo-SMCC-conjugation.jpg]] 

 

=== Synthesis of PHP-TREN-B1 ===

'''10/25/14, Synthesis''' 

1.5 g DAP + 3.6 g DBP + 1.5 ml MeOH, dissolve, feel heat 

+(200 ul TREN + 0.5 ml MeOH) 

mix, 50 oC react o/n 

add 1 ml MeOH, 60 oC react 3 hour 

'''10/26/14 Purification''': + H2O to 10 ml, + 40 ml isopropanol, spin, repeat once. 

Dissolve in H2O to 10 ml 

'''10/30/14, Modification''' 

Make Sulfo-SMCC: dissolve 5 mg Sulfo-SMCC in 50 ul DMSO. Final conc: 0.1 mg/ul 

{| border="1" width="718" cellspacing="1" cellpadding="1"
|-
| Code 
| Name 
| Modification 
| Peptide 
| Peptide Sequence 
|-
| P 
| PHP-TREN-B1 
| 20 ul P 
| 
| 
|-
| S 
| PHP-TREN-B1-Cho     
| 20 ul P+ 5 ul 0.5 ug/ul Cho-Chl 
| 
| 
|-
| T 
| PHP-TREN-B1-Gu
| 20 ul P + ~5 mg pyrozole 
| 
| 
|-
| U1 
| 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 
| 5H-Tat-5H-C 
| HHHHHGRKKRRQRRRPPAGHHHHHC 
|-
| U2 
| 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 
| HA2-R9 
| GDIMGEWGNEIFGAIAGFLGGGRRRRRRRRRGC 
|-
| U3 
| 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 
| RRRQRRKKRGLFEAIEGFIGNGWEGMIDGWYGC 
|-
| U4 
| 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 
| RRRRRRRRRGLFEAIEGFIENGWEGMIDGWYGC 
|-
| U5 
| 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 
| Tat-HA2 
| RRRQRRKKRGGDIMGEWGNEIFGAIAGFLGC, $215/mg 
|-
| H6 
| PHP-TREN-B1-C-5H-Tat-5H-C 
| 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of 5.6 mg/ul peptide 
| C-5H-Tat-5H-C 
| CHHHHHGRKKRRQRRRPPAGHHHHHC 
|-
| U7 
| PHP-TREN-B1-Tat2 
| 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add '''2.5''' ul of '''10.5''' mg/ul peptide 
| Tat2 
| C(YGRKKRRQRRRG)2 
|}

 

'''10/30/14: Transfection''' 

293T, 24 wells, 1 ug DNA, 3 ul diluted TR: 1;30, 1:50, 1:75 

'''10/31/14 Result''' 

[[Image:PHP-TREN-B1 peptide 10 31 2014.png]] 

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 

[[Image:PHP-TREN-B1 peptide 11 2 2014.JPG]] 

 

11/3/14

[[Image:PHP-TREN-B1 peptide 11 3 2014 2.JPG]]

''' '''

'''2nd Transfection''' 

11/2/14

[[Image:PHP-TREN-B1 peptide 11 2 2014 2.JPG]] 

11/3/14

contaimination--> no or few green cells

[[Image:PHP-TREN-B1 peptide 11 3 2014.JPG]]

 

=== Synthesis of PHP-TREN-B1-Cho-Gu-Peptide ===

'''Synthesis''' 

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 

{| border="1" width="718" cellspacing="1" cellpadding="1"
|-
| Code 
| Name 
| Modification 
| Peptide 
| Peptide Sequence 
|-
| Q 
| PHP-TREN-B1-Cho-Gu 
| 20 ul Q PHP-TREN-B1-Cho-Gu, + 10 ul H2O 
| 
| 
|-
| V1 
| 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 
| 5H-Tat-5H-C 
| HHHHHGRKKRRQRRRPPAGHHHHHC 
|-
| V2 
| 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 
| HA2-R9 
| GDIMGEWGNEIFGAIAGFLGGGRRRRRRRRRGC 
|-
| V3 
| 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 
| RRRQRRKKRGLFEAIEGFIGNGWEGMIDGWYGC 
|-
| V4 
| 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 
| RRRRRRRRRGLFEAIEGFIENGWEGMIDGWYGC 
|-
| V5 
| 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 
| Tat-HA2 
| RRRQRRKKRGGDIMGEWGNEIFGAIAGFLGC, $215/mg 
|-
| V6 
| PHP-TREN-B1-Cho-Gu-C-5H-Tat-5H-C 
| 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add 5 ul of 5.6 mg/ul peptide 
| C-5H-Tat-5H-C 
| CHHHHHGRKKRRQRRRPPAGHHHHHC 
|-
| V7 
| PHP-TREN-B1-Cho-Gu-Tat2 
| 2 ul Sulfo-SMCC (200 ug), incubate at RT for 30 min, add '''2.5''' ul of '''10.5''' mg/ul peptide 
| Tat2 
| C(YGRKKRRQRRRG)2 
|}

 

 '''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]]

 

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 

[[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 

[[Image:Q V 2014 11 12.JPG]]

 

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 [[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 [[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 [[Image:Q V 2014 11 13.JPG]]

 

 

11/14/14 

[[Image:Q V 2014 11 14 1.JPG]]

11/15/14 

[[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 

dissolve in 12 ml H2O 

+ 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. 

Dialyse vs water, freeze dry, dissolve dried pellet in 10 ml water. --> '''W ''' 

{| 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 

[[Image:S T W 2014 11 13.JPG]] 

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]]

 

'''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]]

 

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]]

 

== 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]]

 

== 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
|}

 

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]]

 

[[Image:12 12 14 3 s.JPG]]

 

[[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]]

Synthesis of PHP-TREN-B1-Peptide - Revision history

Paul.xu at 23:21, 15 December 2014