How do I determine the degree of conjugation of the immobilized ligand?
Protein samples can be quantitated using Pierce BCA Protein Assay (Product # 23225) or Coomassie Plus Protein Assay Kit Product # 23236 (with this product read absorbance at 465 nm instead of 595 nm)], comparing quenched, unconjugated gel to conjugated gel. You can also compare the change in absorbance at A280 for the proteins before and after coupling. Finally, use Ellman's Reagent to determine the loss of reduced sulfhydryls.
How do I perform affinity purifications with conjugated SulfoLink Affinity Columns?
Equilibrate the column with 3-5 bed-volumes of an appropriate binding buffer. Add 1 ml of sample for each 2 ml column (serum should be diluted at least 1:1 with binding buffer). Add an additional 200 µl of binding buffer to ensure that the entire sample has entered the gel bed. Cap the column bottom and top. Incubate the column for 1 hour. Wash away non-bound proteins with 5-7 bed-volumes of binding buffer or 1 M NaCl. Elute the bound sample by adding small fractions (0.5-1.0 ml) of elution buffer such as Pierce IgG Elution Buffer (Product # 21004).
How do I reduce my sample and confirm the reduction?
The kit includes the reductant beta-Mercaptoethylamine (ß-MEA, Product # 20408); however, the following reductants can also be used: dithiothreitol (DTT), Product # 20290; ß-MEA # 35600 and 35601; and TCEP?HCl, Product # 20490. You can also use Ellman's Reagent (Product # 22582) to confirm the presence of sulfhydryls. Ellman's Reagent reacts with reduced sulfhydryls to produce a distinctive yellow color that is readable at 412 nm. After it is reduced, desalt the sample directly into an appropriate buffer and allow it to react with the column. This will prevent disulfide formation from reforming.
How does SulfoLink Immobilization work?
SulfoLink Resin is crosslinked 6% beaded agarose that has been derivatized with a 12-atom spacer arm that ends in an iodoacetyl group. This spacer arm is helpful for peptides or large proteins that might otherwise be sterically hindered in binding their ligand. The iodoacetyl group reacts with free sulfhydryls at pH 7.5-8.5 to form covalent thioether bonds. The sulfhydryl must be present in the reduced form because disulfides will not react with the iodoacetyl group.
How many purifications can I perform using the same SulfoLink Column?
The columns can be reused at least 10 times without loss of activity.
Must I block the resin before each use?
No. The resin will no longer react with subsequent sulfhydryls after the ligand is attached and the remaining active sites are blocked.
My peptide is not soluble in the coupling buffer. What can I do?
SulfoLink Immobilization is compatible with 3-4 M fresh urea or guanidine. Alternatively, dissolve the peptide in 100% DMSO. Add the peptide in DMSO to the coupling buffer so that the DMSO does not exceed 20% of the final solution.
What is the binding capacity of SulfoLink Resin?
SulfoLink Resin can bind approximately 5 mg of reduced IgG per ml of support. For peptides, the capacity is approximately 1 mg of peptide per ml of gel. Capacity for other compounds will vary.