List of protocols
Aldehyde Functionalisation
CA - DEAE Derivatisation
CA - Epichlorohydrin Activation
EDC NHS Activation
Aldehyde Functionalisation
1. Electrospin cellulose acetate (Mr = 29,000) 0.20g/mL in acetone/DMF/ethanol (2:2:1) @ 800µL/h and 20kV.
2. Treat the subsequent fibres at 210°C for 5 mins
3. Deacteylize CA membrane by treating with 0.1M NaOH (4mg/mL) in H2O/EtOH 2:1 for 24 h to get a regenerated cellulose membrane
4. Oxidize in 0.05M sodium periodate (NaIO4) to produce dialdehyde groups for 6 h in Darkness (0.2g in 20mL PBS OR 50mM = 10.7mg/mL) (NaIO4) Mr = 213.89.
5. To this 20mL solution add 10mg RC membrane. Carry out this oxidization at rt in darkness with gentle shaking
6. Cease this reaction by the addition of ethylene glycol after 6 - 12 hours (5mL) stir for 30 mins
7. Wash with DI H2O Thoroughly
8. Immerse in 5mL rSpA in PBS (1mg/mL) at rt for 12 h
9. Deactivate remaining aldehyde groups by the addition of 1M ethanolamine (in PBS) 24h @ 2°C, Wash in PBS
10. Convert initially formed carbinolamine into more stable amine be adding sodium cyanoborohydride (NaBH3CN 62.84 g/mol) 5mg in 1mL borate buffer. Stirred for 30 mins @ rt. Add additional 5mg and stir for 30 mins.
11. Wash in PBS & DI H2O for 2 h
Use 50% molar excess NaCNBH3
Cellulose Monomer Unit Mr = 162 g/mol Has 3 ‘-OH’ groups per unit
(63/162) x 3 = 1.17
So for 150% excess for 10mg RC membrane use 17.5mg NaCNBH3
OR use 50mM = 3.14mg/mL in 0.1M Borate Buffer pH9
Boric Acid Mr = 61.83. 0.1M = 6.18mg/mL (3.09g in 500mL)
Adjust pH with 4mL 4M NaOH (160mg/mL) (640mg)
ca - DEAE Derivatisation
CA activation with 2-(diethylamino) ethyl chloride hydrochloride (DAECH) to yield DEAE anion-exchange cellulose nanofiber felts.
1. Deacetylation by immersion in a 0.05M NaOH aqueous 24 h
2. Rinse in DI H2O 3 x, dried in a vacuum oven at 60 ◦C
3. Immerse in a 15% (mass fraction) DAECH aqueous solution 10 min, dry at 60 ◦C
4. Immerse 0.5M NaOH aqueous solution at 90 ◦C for 10 min
5. Finally rinse in DI H2O 3 x, dried in a vacuum oven at 60 ◦C
Epichlorohydrin Activation
1. Electrospin cellulose acetate (Mr = 29,000) 0.20g/mL in acetone/DMF/ethanol (2:2:1) @ 800µL/h and 20kV.
2. Treat the subsequent fibres at 210°C for 5 mins
3. Deacteylize CA membrane by treating with 0.1M NaOH (4mg/mL) in H2O/EtOH 2:1 for 24 h to get a regenerated cellulose membrane
4. Wash membrane with DI H2O
5. Add 20mL water and shake at room temperature
6. Add sufficient 10M NaOH to obtain a final concentration of 1.1 M in the reaction. (0.88g)
Set the temperature to 34 °C and heat with shaking until the temperature in the reaction vessel is constant (about 30 min).
7. Add Epichlorohydrin to final conc 2.2M. (92.52 g/mol = 0.2035g/mL = 4.07g)
The temperature should rise to about 40°C upon mixing.
8. Set the temperature to 40 °C and leave reaction shaking for 3h
9. Stop the reaction decanting the reaction mixture and washing the membrane in 10 volumes of distilled water. Wash with DI H2O Thoroughly
10. Immerse in 5mL rSpA in PBS (1mg/mL) at rt for 12 h
11. Deactivate remaining aldehyde groups by the addition of 1M ethanolamine (in PBS) 24h @ 2°C, Wash in PBS
12. Wash in PBS & DI H2O for 2 h
EDC NHS Activation
1. Add 0.4 mg of EDC (final concentration 2 mM) directly to 1 ml of Protein #1 (COOH group), which based on a 50 kDa protein, results in a 10-fold molar excess of EDC to Protein #1.
2. Add either 0.6 mg of NHS or 1.1 mg of Sulfo-NHS to the reaction (final concentration 5 mM). If using the No-Weigh Format of Sulfo-NHS add 40 μl of ultrapure water or Activation Buffer to an individual microtube, which yields 230 mM; then add 22 μl of the dissolved reagent to the 1 ml reaction (final concentration 5 mM).
3. Mix reaction components well and react for 15 minutes at room temperature.
4. (Optional): Add 1.4 μl of 2-mercaptoethanol (final concentration of 20 mM) to inactivate the EDC.
5. (Optional): Separate activated Protein #1 from excess EDC, EDC-byproducts, NHS and (if used) 2-mercaptoethanol using an appropriate size desalting column that has been equilibrated with PBS. Follow desalting column instructions and recover the fraction containing the activated protein. If using absorbance at 280 nm to identify fractions containing protein, be aware that NHS and Sulfo-NHS absorb strongly at 260-280 nm.