Desalting Column Protocol Preparation 1. 2. 3. 4.

Obtain column from fridge and invert column a few times to resuspend the resin. Remove storage solution- dump the azide into the azide waste inside hood Attach to clamp on stand. Obtain sterile water, 10 epindorphs, waste beaker.

Wash the Column 1. 2. 3. 4. 5. Take off bottom cap of column. Add about 3 ml of water to column with pipette. Let drip into waste beaker. Wash for about 1 hour- Add about 10 ml of water total. Things to do while waiting: mark the tubes at ml, number tubes, and obtain sample- record sample information. 6. When finished, place cap back on bottom of column. Running the Column 1. Place sample on top of column and let sample sink into resin. 2. Add ml water to top and start collecting in tubes (lower clamp to allow tube to rest again the end tip of the column. 3. Add ml water again when it stops dripping or runs low. 4. Collect 8-10 tubes. 5. After wash column with water into waste beaker- use about 5 ml water. UV Absorbance Protocol 1. 2. 3. 4. 5. 6. 7. 8. Place 100 ul of product in black cuvette. Wipe cuvette with Chemwipe. Insert cuvette- rough corner in the back. On instrument, click TEST---MULTIWAVELENGTH---SET nm---280 nm, 254 nm, 215 nm. Click RUN TEST Wash and dry cuvette between each test- Rinse with ddH20 and place cuvette on vacuum. Dry with chemwipe. Repeat these steps for all samples and record data.

UV Absorbance -Protein= 280 -Phenylalanine (F) = 254 -Peptide= 215 -Salts should come out at the end-after 8 tubes