Thiolation of EGF and Conjugation to Liposome
Run 1
August 19 Present: Chanpreet, Edward, Kelvin, Tyra
Task: To thiolate EGF, and dialyze the buffers to stop further thiolation
Thawing of reagents
- used our own centrifuge, at default speed setting
- used a counterbalance of 1mL of water in the microcentrifuge tube
- centrifuged Traut’s for 4 minutes, checked on it, and then for another 1.5 minutes
- centrifuged EGF for 4 minutes
- Protocol: Placed the microcentrifuge tube containing the reagent in the centrifuge, placed the counterbalance opposite, turned the centrifuge on, waited the allotted time, turned the centrifuge off
Thiolation
- Pipetted 260uL of the thawed Traut’s into the microcentrifuge tube containing 1mL of thawed EGF
- incubated at room temperature for 1 hour
Dialysis
- washed dialysis tube by pipetting in 2.5mL of millQ, waiting 5 minutes, then dumping out milliQ
- repeated the wash using PBS instead of milliQ
- pipetted in all 1260uL of thiolated EGF into the dialysis tube
- filled a 500mL beaker with 378mL of PBS (300x the volume of the thiolated EGF) (at room temperature)
- pushed the dialysis tube into the styrofoam, and the placed it in the beaker
- started stirring using a stir plate at 60RPM after 15 minutes of no stirring
Note: had to decrease the diameter of the styrofoam floatie so it would fit in the beaker
Dialysis Part 2
- kept some (~100mL) of the waste dialysate, dumped the rest down sink
- washed beaker that dialysis was done in with DI and wiped it down with a kimwipe before refilling it with 378mL of PBS (at 4°C)
- put the dialysis tube back in the beaker to continue second round of dialysis
- stirring using stir plate at 60RPM, to be left overnight
August 20, 2016
Present: Ina, Valerie, Chanpreet, Edward
Goal: Collect dialysis samples for assays and conjugate thiolated EGF to liposomes
Post Dialysis
Noted that the sample absorbed some of the buffer through dialysis
- expected 1260uL, actually had ~1343uL
- took a 126uL sample and a 117uL sample of the dialysate
Conjugation of EGF to liposome
- pipetted 550uL of EGF sample to a microcentrifuge tube
- pipetted 500uL of liposomes to the tube
- repeated once for a total of two 1050uL of EGF-liposome samples
- centrifuged at 6000RPM for 3 seconds to ensure solution is at bottom of tube
- incubating by leaving it at room temperature on lab bench, started at 12:45
- not using orbital shaker
Notes:
- should exect less EGF conjugated to liposomes because EGF sample is more dilute
August 21, 2016
Present: Edward, Valerie
Goal: Take sample of EGF-conjugated liposomes, add B-Mercaptoethanol to the liposomes, and dialyse to remove B-Mercaptoethanol
Sampling EGF conjugated liposomes
- Pipetted 50uL from each of the two microcentrifuge tubes containing the EGF-conjugated liposomes into a microcentrifuge tube
- Centrifuged the collected sample at 6000RPM for 3 seconds
- Labelled and stored at –20°C
Adding B-Mercaptoethanol
- all procedures done in fume hood
- pipetted 200uL of B-Mercaptoethanol into a microcentrifuge tube as stock
- pipetted 43uL from the stock into each of the two microcentrifuge tubes containing the EGF-conjugated liposomes
- centrifuged at 6000RPM for 3 seconds
- incubated at room temperature on lab bench
Dialysis of B-Mercaptoethanol
Part 1
- washed dialysis tube by pipetting in 2.5mL of millQ, waiting 5 minutes, then dumping out milliQ
- repeated the wash using PBS instead of milliQ
- pipetted in all 2086uL of liposome-EGF-Beta-mercapotoethanol solution from both microcentrifuge tubes into the one dialysis tube
- filled a 500mL beaker with 420mL of 4 degree C PBS (~200X volume ratio)
- pushed the dialysis tube into the styrofoam, and the placed it in the beaker
- started stirring using a stir plate at 60RPM
- not dialysing in the fume hood because the sample is not releasing any smell
Part 2
- kept some (~100mL) of the waste dialysate, poured rest into waste flask on bench
- washed beaker that dialysis was done in with DI and wiped it down with a kimwipe before refilling it with 420mL of PBS (at 4°C)
- put the dialysis tube back in the beaker to continue second round of dialysis
- stirring using stir plate at 60RPM, to be left overnight
Since we used the dialysis tubes, any unbound EGF will still be in the final sample
August 22, 2016
Task: Transferred dialysate from dialysis tube into microcentrifuge tubes
Final
- pipetted 1 mL of final EGF-conjugated liposomes into each of 2 microcentrifuge tubes
- pipetted the remaining ~96.6uL into a third microcentrifuge tube
- labeled and stored at –20°C for 1.5 hours, then moved to 4°C
- sample increased by ~10uL in volume due to dialysis, shouldn’t affect concentration too much
Run 2
Why are we doing this?
- Did DLS on the final Liposome sample didn’t get good results
- Mina said that freezing the liposomes damaged them, and basically our sample is unusable
August 23, 2016
Task: Thiolation of EGF and Dialysis
Thawing of reagent
- used a counterbalance of 1mL of water in the microcentrifuge tube
- centrifuged 1 mL of 368.41uM of Traut’s for 4 minutes at 6000RPM
- centrifuged 1mL of 23.87uM EGF for 4 minutes at 6000RPM
- Protocol: Placed the microcentrifuge tube containing the reagent in the centrifuge, placed the counterbalance opposite, turned the centrifuge on, waited the allotted time, turned the centrifuge off
Thiolation
- Pipetted 260uL of Traut’s Reagent into the microcentrifuge tube containing the EGF
- incubated for 1 hour at room temperature
Dialysis
- washed dialysis tube by pipetting in 2.5mL of millQ, waiting 5 minutes, then dumping out milliQ
- repeated the wash using PBS instead of milliQ
- pipetted in all 1260uL of thiolated EGF into the dialysis tube
- filled a 500mL beaker with 378mL of 4°C PBS (300x the volume of the thiolated EGF)
- pushed the dialysis tube into the styrofoam, and the placed it in the beaker
- stirred using a stirplate at 60RPM for 1.5 hours, then left it without stirring for another 0.5 hours
Dialysis Part 2
- replaced the dialysis buffer with a fresh 400mL of 4°C PBS
- kept a sample of the waste, dumped the rest down the drain
Post Dialysis
- kept a sample of the wste buffer, dumped the rest down the drain
- pipetted 550uL each into 2 microcentrifuge tubes
- found that there was an additional 284 uL of sample remaining (expected 160uL remaining)
- pipetted an additional 50uL of sample each into the 2 tubes
- to find the amount I needed to keep the number of mols of EGF the same: 550/1260*(1384) ~= 600
- kept the remaining 184uL of sample in –20 freezer in red box labelled as T2
Conjugation of EGF to liposomes
- added 500uL each of liposomes into the 2 tubes containing the thiolated EGF
- labelled the tubes on the lid as L’
- for one tube containing EGF, additionally labelled 488, there was only 488uL of sample in one of the liposome tubes
- pipetted the 488uL into the tube containing the EGF, then topped up the liposomes by pipetting 10uL then 2uL from the liposome tube labelled as S
- for the other tube, could only pipetted 498uL, topped up the remaining 2uL of liposomes from the liposome tube labelled as S
- centrifuged both tubes at 6000RPM for 3 seconds to ensure solution is at bottom of tube
- incubating overnight at room temperature, started at 2:30Pm Aug 24
August 25, 2016
Removal of EGF from liposomes
- used 300kDa dialysis tubing
- cut open the bag containing the dialysis tubing
- cut ~8cm of tubing, put remaining back in and sealed bag with tape
- soaked tubing in milliQ for 15 minutes
- rinsed tubing with 3mL of milliQ then 3mL of PBS
- pipetted 2mL of sample into tubing
- spilled some sample, now left with 1.48mL
- used 465mL of PBS for dialysis
- tried to use a jar for dialysis, didn’t really quite fit properly so Ina got a 500mL beaker
Dialysis Part 2
- had to make more PBS (ran out)
- see preparation of buffers
- made 500mL of 1X PBS from the 10X PBS
- added 50mL of 10X PBS to a 500mL graduated cylinder
- adjusted volume to 500mL
- adjusted pH to 7.48
- replaced dialysis buffer with a fresh 465mL of PBS (homemade pH 7.48)
- kept a sample of waste buffer for testing, stored rest in a flask on the bench (labelled as EGF-liposome dialysis waste)
Aug 26, 2016
Finishing up Dialysis
- was only ~1050uL of sample after the dialysis
- somehow there as nearly 500uL loss in sample
- not sure if the tube was leaking and we lost liposomes or just buffer
- pipetted 750uL and 300uL into two microcentrifuge tubes, lablled F2 in the 4°C fridge
Run 3
Task: thiolation of EGF and dialysis
- Centrifuged EGF for 4 minutes at 6000RPM with a 1mL water counterbalance
- Pipetted 65uL of Traut’s into reagent containing EGF and incubated at room temperature for one hour
Dialysis
For dialysis, total buffer (PBS) used is 350 mL
Dialysis Part Two
After two hours, buffer changed with another 350 mL and left overnight
Conjugation of EGF to Liposomes
- Added 125 uL liposomes to 137.5 uL EGF twice (in 2 separate microcentrifuge tubes) for total of 535 uL (note: this is 1/4 of what was previously done)
- Centrifuged at 6000 rpm for 3 s and incubated at room temperature overnight
Task: dialysis of unbound EGF to liposomes
- Buffer used was 400 mL (1000x the EGF) of PBS because 200 mL was too small to be fully submerged in the beaker and not hit the stir bar