How do AminoLink Resins and Kits work?
Both AminoLink Coupling Resin and AminoLink Plus Coupling Resin consist of a crosslinked beaded agarose that is derivatized to have aldehydes present for coupling. The aldehydes will react with primary amines to form reversible Schiff?s bases. After this double bond reacts with sodium cyanoborohydride, the process of reductive amination produces a permanent covalent bond. The aldehyde groups attach directly to the beads without any spacer arms.
How do I determine the degree of conjugation of the immobilized ligand?
Protein samples can be quantified using Pierce BCA Protein Assay (Product # 23225) or Coomassie Plus Protein Assay (Product # 23236).
How can I perform affinity purifications with conjugated AminoLink and AminoLink Plus Affinity Columns?
Equilibrate the prepared column with three to five bed volumes of an appropriate binding buffer. Add 1 ml of sample for each 2 ml bed-volume (serum should be diluted at least 1:1 with binding buffer). Add an additional 200µl of binding buffer to ensure that the entire sample has entered the gel bed. Cap the column bottom and top. Incubate one hour. Wash away non-bound proteins with five to seven 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 long will the coupling reaction take?
The immobilizatoin reaction takes six hours; the entire procedure may require seven to eight hours. This time can be decreased if the high pH protocol for AminoLink Plus Resin is used.
How many purifications can be performed using the same AminoLink or AminoLink Plus Column?
Columns can be reused at least 10 times without loss of activity.
My ligand is not soluble in the coupling buffer. What can I do?
Coupling reactions with AminoLink Resins are compatible with 3-4 M fresh urea or guanidine. (However, old solutions of urea may contain ammonia, which will interfere with ligand immobilization.) Alternatively, dissolve the peptide in 100% DMSO. Add the peptide in DMSO to coupling buffer so that the DMSO does not exceed 20% of the final solution.
What are the differences between the two supports?
The AminoLink Plus Resin uses a type of agarose that has better flow properties, which may shorten the time needed for drip-column methods.
What is the binding capacity?
AminoLink Coupling Resin can bind approximately 10 mg of IgG/ml of resin bed, while AminoLink Plus Resin can bind up to 15-20 mg of IgG/ml of resin bed. The upper limit for binding peptides is approximately 1 mg/ml of resin for both support types.