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View additional product information for SulfoLink™ Immobilization Kit for Peptides, 2 mL - FAQs (44999)
8 product FAQs found
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.
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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.
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No. The resin will no longer react with subsequent sulfhydryls after the ligand is attached and the remaining active sites are blocked.
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The columns can be reused at least 10 times without loss of activity.
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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.
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The kit includes the reductant beta-Mercaptoethylamine (beta-MEA, Cat. No. 20408); however, the following reductants can also be used: dithiothreitol (DTT), Cat. No. 20290; ß-MEA Cat. No. 35600 and 35601; and TCEP-HCl Cat. No. 20490. You can also use Ellman's Reagent (Cat. No. 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.
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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 Thermo Scientific IgG Elution Buffer (Cat. No. 21004).
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The cysteine sulfhydryl is very labile when exposed to air or in solution, and it can easily oxidize with another sulfhydryl. This typically happens between sulfhydryls from two molecules, but it can sometimes occur if there are at least two cysteines present on the same peptide or protein. This oxidation forms a disulfide bond and changes the two cysteines into cystine.
TCEP is one of three commonly used reducing agents that can break a disulfide bond. The other common reducing agents are beta-mercaptoethanol (β-ME or 2-ME) and dithiothreitol (DTT).
In step A-3 of the manual, one would carry out the TCEP reduction by adding 0.1 mL TCEP to the solution prepared in step A-1 and let incubate for 30 minutes. After the SulfoLink Resin is prepared in steps B-1 to B-3, one would add the sulfhydryl-containing molecule (result of step A-3) to the SulfoLink Resin.
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