Periodate Oxidation of APOE
Trial 1:
September 24, 2016
Present: Lily, Ina
Task: Oxidation of APOE and conjugation of oxidized APOE to DSPE-PEG-CONHNH2
From last session: we left APOE in the –20 after doing a wash with PBS and 1% BSA, we assume that we have 0.05 mg of APOE in the tube.
- Resuspended 0.05 mg APOE in 100 uL oxidation buffer to get a final concentration of 0.5 - 1 mg/mL
- In a dark room, weighed out 2.1 mg of Na-periodate and dissolved in 500 uL of oxidation buffer
- Added 400 uL oxidation buffer to the 100 uL APOE suspended in oxidation buffer for a total volume of 0.5 - 1 mg/mL
- Note: Added 5 uL of 40 mM sodium borohydride too quickly! Did not wait 10 minutes, we will try again waiting 10 minutes for the reaction to occur first in trial 2.
- After 10 minutes oxidized APOE was purified using gel filtration chromatography
Conjugation:
- Original concentration of DSPE-PEG-CONHNH2: 10 mgmL, performed a 10 fold dilution (2 uL of DSPE-PEG-CONHNH2 + 18 uL of dH2O to get 0.1 mg/mL)
- DSPE PEG = 1 mg/mL, to get a ratio of 12:1 APOE:DPC we add 4.2 uL of DSPE-PEG-CONHNH2 + 95.8 uL dH20 and 100 uL oxidized APOE (0.5 mg/mL)
- Incubated for 2 hours at room temperature
- Product was filtered and then assessed using MALDI-MS
Trial 2:
October 13, 2016
Present: Lily, Ina
Task: Oxidation of APOE and conjugation of oxidized APOE to DSPE-PEG-CONHNH2
- Resuspended 0.05 mg APOE in 100 uL oxidation buffer to get a final concentration of 0.5 - 1 mg/mL
- In a dark room, weighed out 2.2 mg of Na-periodate and dissolved in 500 uL of oxidation buffer
- Added 400 uL oxidation buffer to the 100 uL APOE suspended in oxidation buffer for a total volume of 0.5 - 1 mg/mL
- Allowed APOE and Na-periodate to react for 10 minutes in the dark
- Next, added 5 uL 40 mM sodium borohydride
- Purified oxidized APOE product using gel filtration chromatography
- The final volume of this was 200 uL
Conjugation:
- DSPE-PEG concentration: 1 mg/mL
- APOE concentration: 0.05 mg/0.200 mL = 0.25 mg/mL
- Since we want to have a 12:1 ratio of APOE:DSPE-PEG-CONHNH2 we diluted DPC to 0.021 mg/mL
- DPC dilution: 1 mg/mL * (X) = (0.021) (1 mL) ; X = 21 uL ; Add 21 uL DPC + 979 uL dH2O
- Added 200 uL of dilution to 200 uL of APOE
- Repeated with a 6:1 ratio as well
- Allowed for incubation for 2 hours at room temperature
- Note: samples were allowed to incubate for 3.5 hours
- 10 kDa amicon centrifuge filters were first washed with 400 µL of milliQ water (12 RCF for 5 mins)
- Since only 1 kDa centrifuge filter was left, only the 12:1 sample was concentrated and purified. 400 µL of 12:1 sample was loaded into the filter and centrifuged for 15 minutes at 12 RCF.
- Immediately after spinning down, the filter tube was reversed in a new collection tube in order to collect the concentrated sample.
- The current sample is at around 0.25 mg/mL in 200 uL. The volume of the sample collected after reverse spinning is around 36 uL. Therefore, the concentration of the collected sample is around 1.38 mg/mL
- 14 uL of milliQ H20 was added to the 36 uL of 1.38 mg/mL to get a final volume of 50 uL and a final concentration of 1.0 mg/mL
- Samples were stored in the 4 deg. C fridge (in box labelled Lily-APOE)