Nectagen

Product Information

General nanoCLAMP(Resin) product information, v 1.0

Protocol for one-step purification from E.coli lysates

1. Transfer 100 µl of 50% slurry of nanoCLAMP(Resin) to a small chromatography column and wash resin three times with 500 µl PBS.
2. Add cleared lysate containing the nanoCLAMP antigen, cap top and bottom of column, and incubate at 4°C rotating for 1 h. Lysates should be cleared of insoluble material by centrifugation, at near neutral pH (Resin tolerates detergents such as those found in BPER (Thermo) and RIPA lysis buffer) Note: nanoCLAMPs typically bind between 20 and 250 nmol/ml resin, depending on antigen and binding conditions. See certificate of analysis for lot-specific measured binding capacity.
3. Drain flow through (do not let resin run dry).
4. Wash resin four times with 500 µl PBS or PBS-T (up to 0.05% Tween 20).
5. Elute resin with 100 µl PEB four times, or until protein is completely eluted.
6. To regenerate column, wash four times with 500 µl DB, then refold nanoCLAMP by washing two times 500 µl MBS, then wash four times with MBS + 1 mM CaCl2.  Add azide to 0.05% and store at 4°C.

Materials Required

• Disposable 1 - 2 ml chromatography column with top and bottom caps
• Phosphate buffered saline (PBS): 20 mM NaH₂PO₄, 150 mM NaCl, pH 7.4
• PBS-T: PBS supplemented with 0.05% Tween20
• E.coli lysate, cleared of insoluble material by centrifugation, at near neutral pH (CR tolerates detergents such as those found in BPER (Thermo) and RIPA lysis buffer)
• Polyol elution buffer (PEB): 10 mM Tris, 1 mM EDTA, 0.75 M (NH₄)₂SO₄, 40% propylene glycol, pH 7.9
• Denaturing buffer (DB): 6 M guanidine-HCl, 100 mM NaH₂PO₄, pH 8
• Mops buffered saline (MBS): 20 mM MOPS, 150 mM NaCl, pH 6.5
• Sodium azide

Product References

This product (“Product”) is warranted to perform in conformance with this Product specification and to be free from defects in material or workmanship for up to one year from time of sale.  This product is for research use only.  Nectagen is under no obligation to replace products as the result of accidents, misuse, disaster, force majeure, fault or negligence of purchaser, use of product in a manner unintended for its use, or improper storage and handling.  No claim of suitability for FDA regulated applications is made.