Synthesis of PHP-TREN-B1-Peptide

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Peptides

[edit]
Name
 Seq
 Form
 Transfection Efficiency
Tat48–60 GRKKRRQRRRPPQ

Tat49-60
 RKKRRQRRRPPQ

Tat47–57
 RKKRRQRRR-GGG

Tat2
 C(YGRKKRRQRRRG)2

HIV Rev
 TRQARRNRRRRWRERQR

Penetratin (Antp 43–58 ) RQIKIWFQNRRMKWKK

Transportan GWTLNSAGYLLGKINLKALAALAKKIL

TP10 AGYLLGKINLKALAALAKKIL

Oligoarginine (R8) RRRRRRRR

MAP KLALKLALKALKAALKLA

MPG GALFLGFLGAAGSTMGAWSQPKKKRKV

MPGa GALFLAFLAAALSLMGLWSQPKKKRKV

CADY
 GLWRALWRLLRSLWRLLWRA-cya

LK15
 RKKRRQRRRGGGKLLKLLLKLLLKLLK-CONH2

KALA
WEAKLAKALAKALAKHLAKALAKALKACEA

Pep1 (Chariot)
 KETWWETWWTEWSQPKKKRKV

INF-7
 GLFEAIEGFIENGWEGMIDGWYG

GALA-INF3 GLFEAIEGFIENGWEGLAEALAEALEALAA

HA2 GLFGAIAGFIENGWEGMIDGWYG

Tat
 RKKRRQRRRPPQ
4X106
Tat-CTHD
 RKKRRQRRRPPQC
 C-Terminal disulfide homodimer
 1.2X107
Tat-NTHD
 CRKKRRQRRRPPQ
 N-Terminal disulfide homodimer
 1.0X107
Cyclic-Tat
 CRKKRRQRRRPPQC
 Disulfide bond between C-terminal and N-terminal
 3.8X107


Synthesis PHP-TREN-B1-Peptide

[edit]

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



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

Sulfo-SMCC



Synthesis of PHP-TREN-B1

[edit]

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

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


11/1/14

11/2/14



11/3/14


2nd Transfection

11/2/14


11/3/14

contaimination--> no or few green cells


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

[edit]

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

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

contamination-->no blue cells

11/3/14


11/7/14

Transfection: ~60% cell density

resulted a lot of cell death because lower cell density

take pictures on 11/13/14

1:30 1:50 1:75 1:30 1:50 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

11/14/14

11/15/14

1:30 1:50 1:75 1:30 1:50 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


11/14/14

11/15/14

1:30 1:50 1:75 1:30 1:50 1:75
S S S T T T

11/13/14

11/14/14

11/15/14

Transfection again on 7/12/14 with higher cell density

picture on 7/13/14

1:30 1:50 1:75 1:30 1:50 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

11/14/14

11/15/14

11/16/14

1:30 1:50 1:75 1:30 1:50 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



11/14/14

11/15/14

11/16/14

Synthesis of PHP-TREN-B1-PEI18-Cho: W

[edit]

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

1:30 1:50 1:30 1:50 1:30 1:50
S S T T W W

11/13/14


11/14/14

11/15/14

11/16/14


New Transfection. 2014/11/19

P, U1 to U7

2014/11/20

2014/11/21

2014/11/22


Q, V1 to V7

2014/11/20

2014/11/21

2014/11/22

S,T, W, Turbofect, L2K, B1

2014/11/20

2014/11/21

2014/11/22


Transfection using Several Cell Lines

[edit]

293E

[edit]

Date:

11/27/14, P, U1~U7, 1:30, 1:50, 1:75

11/27/14, Q, V1~V7, 1:30, 1:50, 1:75

11/27/14, W, P, Q, R, X, L2K

Hela

[edit]

Transfection Date: 11/25/14

Picture of 11/27/14

Q, V1 ~V7, 1:30, 1:50, 1:75

11/30/14

11/27/14, P, U1~U7, 1:30, 1:50, 1:75

11/30/14

11/27/14, W (1:30, 1:50, 1:75), L2K, X, P, Q (1:30, 1:50), R (1:30, 1:50), B1

11/30/14


Transfection of CHO cells

[edit]

Transfection reagents: P, Q, R, W, 1 ug DNA

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

plate 1,



2014/12/15

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