APOE-only Liposome/APOE + EGF Liposome Protocol
October 14, 2016
Preparation of APOE
- Pre-rinsed the 10 kDa Amicon Centrifuge Filter to remove trace amounts of glycerin by adding 400 uL of Milli-Q water
- Spin tube for 5 minutes at 10,000 rcf and discard water
- From the 50 ug APOE vial, transferred the complete volume into the 10 kDa centrifuge filter (volume around 45 uL since concentration approximately 1.1 mg/mL
- Sigma Aldrich
- Product: SRP6303-50UG
- Lot: 2H177284
- Rinsed out the original APOE vial with 100 uL of PBS and transferred the volume to the centrifuge filter
- Gibco PBS pH 7.4 (1x)
- Product: 10010-023
- Lot: 1806080
- Counter balance the filter and spin at 14,000 rcf for 5 mins
- Approximately 50-80 uL of liquid observed remaining
- Top up with 100 uL of PBS into the centrifuge filter and repeat spin at 14,000 rcf for 5 mins
- Repeated for 2 more cycles to complete buffer exchange
- Inverted the tube into a clean new tube and spin for 2.5 min at 4000 rpm to ensure transfer of liquid
- Kept the centrifuge filter to use after thiolation (this will minimize and losses)
Thiolation of APOE (performed in parallel with Thiolation of EGF, see below)
- Added 16 uL of 368.41 uM 2-iminothiolane solution to the tube containing the APOE in PBS
- Gently mixed and incubate at room temperature for 1 hour
- Transferred the tube contents back into the 10 kDa centrifuge filter via pipette
- Add 100 uL of PBS and spin down using above procedure
- Counter balance the filter and spin at 14,000 rcf for 5 mins
- Top up with 100 uL of PBS into the centrifuge filter and repeat spin at 14,000 rcf for 5 mins
- Inverted the tube into a clean new tube and spin for 2.5 min at 4000 rpm to ensure transfer of liquid
- Final measured volume: 70 uL
- Volume split into 2 x 35 uL aliquots
- One aliquot used for APOE-only liposome
- Second aliquot used for APOE and EGF liposome
- Volume split into 2 x 35 uL aliquots
Thiolation of EGF (performed in parallel with Thiolation of APOE, see above)
- Added 61.6 uL of 23.87 uM EGF stock solution in PBS (prepared on Sept 18, 2016) to a new centrifuge tube
- Amount of EGF calculated based on same moles of APOE
- Added 16 uL of 368.41 uM 2-iminothiolane solution to the tube containing the EGF in PBS
- Gently mixed and incubate at room temperature for 1 hour
- Transferred the tube contents into a new 10 kDa centrifuge filter via pipette
- Add 100 uL of PBS and spin down using above procedure
- Counter balance the filter and spin at 14,000 rcf for 5 mins
- Top up with 100 uL of PBS into the centrifuge filter and repeat spin at 14,000 rcf for 5 mins
- Inverted the tube into a clean new tube and spin for 2.5 min at 4000 rpm to ensure transfer of liquid
- Filtrate was also collected
- Final measured volume: 62 uL
- Volume split into 2x 31 uL aliquots
- One aliquot used for APOE and EGF liposome
- Second aliquot for storage
- Volume split into 2x 31 uL aliquots
October 15, 2016
Note: it was later realized that the 10kDa centrifuge filter has a too large pore size for the 6.2 kDa EGF. Losses of EGF in the filtrate.
Conjugation of APOE with Liposomes for APOE-only Liposomes
- Added 44.8 uL of 18.9 mg/mL liposome solution to the 35 uL aliquot of thiolated APOE
- Incubate at room temperature overnight (9:30 pm start)
- Finish conjugation reaction at 3:30 pm (18 hours incubation with APOE Solution)
- Tube contents were transferred to centrifuge filter via pipette
- To match volume and counter balance with APOE EGF liposome (below), 200 uL of PBS was added
- Spin for 5 mins at 14,000 rcf
- Volume decreased to ~ 200 uL after spinning so continued to spin for an additional 5 mins at 14,000 rcf
- Liposomes aggregation and fouling on the membrane observed
- Centrifuge filter was washed with 100 uL of PBS and centrifuged at 14,000 rcf for 5 mins
- Tube inverted into new tube and fraction was collected by spinning at 1,000 rcf for 2.5 mins
- Final product stored at 4°C
- Final Volume: 110 uL
Conjugation of APOE and EGF with Liposomes for APOE and EGF Liposomes
- Combined 35 uL aliquote of thiolated APOE with the 31 uL aliquot of thiolated EGF
- Added 44.8 uL of 18.9 mg/mL liposome solution to the thiolated protein solution
- Incubate at room temperature overnight (9:30 pm start)
- Since losses may have occurred in the EGF amount due to filter selection, the second 35 uL EGF aliquot was added to the liposome and protein solution at 12 pm (Oct 15th, 2016), as well as 200 uL of the centrifuge filtrate (approx. half of the filtrate)
- More EGF conjugated onto the liposome is not a problem, but 2-iminothiolane will bind to the maleimide sites as well
- At max 6% EGF instead of original 3.5% EGF onto liposome
- At max 18% 2-iminothiolane binding onto liposome
- More EGF conjugated onto the liposome is not a problem, but 2-iminothiolane will bind to the maleimide sites as well
- Finish conjugation reaction at 3:30 pm (14.5 hours with APOE/EGF Solution, 3.5 hours with EGF filtrate)
- Tube contents were transferred to centrifuge filter via pipette
- Spin for 5 mins at 14,000 rcf
- Volume decreased to ~ 200 uL after spinning so continued to spin for an additional 5 mins at 14,000 rcf
- Liposomes aggregation and fouling on the membrane observed
- Centrifuge filter was washed with 100 uL of PBS and centrifuged at 14,000 rcf for 5 mins
- Tube inverted into new tube and fraction was collected by spinning at 1,000 rcf for 2.5 mins
- Final product stored at 4°C
- Final Volume: 110 uL