Procedure to label collagen with Sulfosanpah
Materials Needed:
Collagen (Sigma)
Sulfo-sanpah (Pierce Chemical)
DMSO
dialysis tubing (MW cutoff 10K; i.e. so 350K collagen won't go through pores)
(Millipore is a possible vendor)
4 L container and stirbar
acetic acid
triethyl amine (TEA)
Background:
This procedure will label the lysine-e-amino group on collagen. The pKa of this group is approximately 10 (S. Wong Chemistry of Protein Conjugation and Cross-linking, p. 13, Table 1). Thus, at pH > 10, the NH3+ will be de-protonated and is a better nucleophile and can then attack the sulfo-SANPAH. However, at pH > about 7 (correspondence with M. Nugent, Sigma, Molecular Probes and USB), the collagen will precipitate out of solution and it will be difficult to label a ‘chunk’ of protein.
Collagen solubility in aqueous solution may be a problem and thus requires organic solvent for the reaction (e.g. DMF or DMSO). We observed that collagen in the presence of sulfo-SANPAH will clump up and also will aggregate in the presence of dye. After the collagen has been labeled, the solubility of collagen is enhanced.
This procedure is modified from Molecular Probes method to produce heavily labeled (DQ) collagen. Contact: Robert Archer ( 541-465-8364) at Molecular Probes.
Reference: Anal. Biochem. (1997) v. 251, pp. 144-152
This reference describes a protocol to heavily label proteins with a dye.
Procedure: (will label 1-5 mg of protein)
1. Dissolve 1-5 mg of collagen in DMSO or DMF. We need to determine which solvent works better. DMF will spontaneously degrade into amines, so it will be better to use DMSO.
2. React for 2-6 h dye in 10 to 20M excess dye to protein at 4 degC on a shaker. (Note: For DQ, MolecProbes uses 55M excess). In order to help the reaction to deprotonate the amines on collagen, add a small bit of TEA (1 mM).
3. For the dialysis, do in coldroom in Zhiping Weng's lab (44 Cummington 2nd floor). (Contact: Xia Hui.) Dialyze the solution to remove the organic solvent and free dye against 4L of acetic acid. The dialysis tubing needs at least 5 to 10 ml of solution, so plan accordingly.
4. Change the acetic acid solution every 4 hrs. After the 4th change of solution, let the dialysis run overnight.
5. Can check to see if the labeling reaction occurred by exposing solution to 350nm and seeing if it will precipitate out.