A. Protocol adapted from C.P. Pathak at Sulzer Biologics

1. Dissolve 10 mg HA in 1.5 ml MES buffer, pH 5.5-6.0.
2. Dissolve 2.5 mg N-hydroxysuccinimide(Sulfo-NHS) in HA/MES solution.
3. Add 1 mg peptide.
4. Add 2-3 mg EDAC and mix well.
5. Mix at 4 C O/N.
6. Precipitate solution in acetone.
7. Redissolve in ddI and lyophilize.
8. Use a protein assay to measure the peptide/HA ratio.

B. Protocol adapted from Pierce protocol for NHS/Sulfo-NHS

1. Add 0.4 mg EDC (~2 mM) and 0.6 mg NHS (~5 mM) to 1 ml of 1 mg/ml HA solution and react for 15 minutes at room temperature.
2. Add 1.4 μl 2-mercaptoethanol (final concentration 20 mM) to quench EDC.
3. Add RGD to the reaction mixture in an equi-molar amount to HA.
4. Allow to react for 2 hours at room temperature.
5. Quench reaction by adding hydroxylamine at final concentration of 10 mM (regenerates original carboxyls) or 20-50 mM Tris, lysine, glycine or ethanolamine (results in modified carboxyls).

C. Combo CP/Pierce protocol:

1. Dissolve 10 mg HA in 1.5 ml MES buffer (0.1 M MES, 0.5 M NaCl, pH 5.5).
2. Add 2.5 mg EDC (~8.7 mM), 2.5 mg NHS (~14 mM) and react for 15 minutes at room temperature.
3. Add 10.6 μl 2-mercaptoethanol (~87 mM) to quench EDC.
4. Add RGD to reaction mixture.
5. React for 8 hours at 4 C.
   6. Quench reaction by adding 9.79 mg glycine (10x molar ratio to EDC).
6. Precipitate in acetone.
7. Redissolve in ddI and lyophilize.
8. Use a protein assat to measure the peptide/HA ratio.

D. Protocol adapted from Rowley, Madlambayan, Mooney. Biomaterials 1999;20(1):45-53

1. Make 1% HA in 0.1M MES buffer, pH 5.5-7.5, 0-0.7M NaCl (Rowley: pH 6.5, 0.3M NaCl)
2. Add NHS or sulfo-NHS at 1:1 molar ratio to EDC, mix 5 minutes
3. Add EDC as percent of -COOH available (10, 20, 50%), mix 5 minutes
4. Add GRDGY after 5 minutes, input 0.1-100 mg peptide/g HA
5. Purify (precipitation or dialysis)

E. Calculations and set-up from 2/02 (Exp VIII, p. 83-89)

1. Dissolve 9 vials: 20 mg GMHA in 2 ml MES buffer (varied pH but not NaCl, see below)
2. x % activation = x mol EDC/mol GMHA disaccharides; 1:1 molar ratio NHS to EDC Separately, make 5 ml 50 mg/ml EDC (0.25 g EDC), 5 ml 30 mg/ml NHS (0.15 g NHS)