LMW Alginate Dialysis and Lyo

6/3/14: LMW Alginate Dialysis and Lyo

Friday, May 30, 2014 3:20 PM

Katrina Wisdom

Objective: Start with unfiltered alginate (that has polyphenols and other impurities/alginate fragments) and end with purified, sterilized alginate that can be reconstituted in DMEM and used in experiments with cells.

Materials

• 8MRAD unfiltered (light brown powder) • DI water • Stir bars • Glass Beakers (2) • Large Nalgene Plastic Beaker • Parafilm • Dialysis tubing • Plastic tubing clamps • Charcoal • Steriflips • Glass lyophilizer tubes

Protocol

1.Resuspension in DI water

a.In -80 freezer, 8MRAD (unflitered) is light brown powder. Take out and let warm to room temperature (Tube labeled '8MRAD LF 20/40')

b. Open tube and measure out 2g.

c. Using a cleaned stir bar and beaker, reconstitute the 2g in 200 mls of DI water.

d. Cover with parafilm (the top of the beaker) to block potential contaminants from coming in.

e. Let stir/ dissolve after a few hours (overnight if necessary, but no longer than this).

f. Once completely dissolved, can start the dialysis. 2.

Dialysis

a.Soak dialysis tubing in slightly warmed water in a big glass beaker (comes in a bath of sodium azide).

b.Get LARGE nalgene plastic beaker - fill to 4L. i. CAUTION: if RGD Alginate reconsituted in Buffer, USE ANOTHER PROTOCOL!

c. Use a small stir bar (not too big) so that it won't knock off the clamps

d. Prepare tape label for 5 washes

e. Clamp tubing about 1 cm from the edge.

f. Pour alginate into tubing - either by hand or with pipette i. Leave room to expand (a good 3-4 inches), as there will be swelling

g. Clamp top of tubing and drape over the side of the Nalgene

h. Make sure the tubing/alginate is not touching the bottom of the Nalgene beaker.

i. Do 5 washes (about 3 per day or so until 5)

j. Put extra tubing in falcon tube in water, and put back in the fridge (as this can be used in the future).

3.Charcoal Filtering (6/5/14)

a.Gently remove the clips and pour the alginate into a beaker (have the tubing dangling over a beaker when the clips are removed. Experiment Notes Page 1 beaker when the clips are removed.

b. Put stir bar in beaker.

c. For every 2 g of alginate, add 1 g of Charcoal. Let stir for a half hour.

d. Then let settle for half an hour once done stirring.

4.Sterile Filtering

a. Label the steriflips

b. Sterile filter this solution in the sterile hood using a 500 ml sterile filtration unit.

c. Transfer to the steriflips after alginate solution is sterilized.

d. Put lid on (lid that has spigot on it) and cool the falcon tubes before lyophilizing.

5. Freezing

a. Refrigerate first in the lab fridge

b. After cooled, transfer to the lab freezer (don't freeze right away to prevent cracking)

c. Refrigerate and freeze in a container that does not constrict the falcon tubes (to prevent cracking)

6. Lyophilizing

a. In the glass lyophilizer tubes, add max of 3 falcon tubes of frozen alginate (alternating in direction)

b. Carefully wedge the top of the glass lyophilizer tube into the fitting on the lyophilizer. Be careful not to pull on the tube fitting on the lyophilizer; hold here securely.

7. Turning off lyophilizer

a. Push button to turn off "vacuum" - green button on lyophilizer

b. Release valve on the tube being released.

c. Take tubes out.

d. Turn off lyophilizer.

e. Turn off vacuum pump.

f. Twist valve to release pressure on top of the lyophilizer.

g. Take off the lid of the lyophilizer.

8. Storing alginate

a. Store in the freezer - they are in the lab freezer. NOTE! Less cracking when put in the fridge first and THEN put in the freezer. Remember this for next time.

Experiment Notes Page 2