Obtained intermediates as well as antibody-dye conjugates were purified using NAP columns from Cytiva Lifescience (Marlborough, MA, USA) pre-packed with Sephadex G-25 Grade DNA resin. Final conjugates were purified on an ÄKTA pure 25 chromatography system equipped with a Superdex 200 Increase 10/300 GL column from Cytiva Lifesciences. Elution was done with PBS pH 7.4 at a flow rate of 0.75 mL/min and fraction size of 0.5 mL. Detection was done photometrically, measuring the absorbances at 280 nm (antibodies) and 495 nm (Fam, Vio515).
Nap columns
Manufactured by Cytiva
Sourced in United States
NAP columns are size-exclusion chromatography devices used for desalting and buffer exchange of protein samples. They are designed to remove low molecular weight substances from protein solutions while retaining the protein of interest.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using nap columns
1
Purification of Antibody-Dye Conjugates
2
Fluorescent Labeling of KSHV Virions
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For fluorescent staining of KSHV, the virus particles were first labeled with 1,2-distearoyl-sn-glycero-3-phosphoethanolamine-N-[methoxy(polyethyleneglycol)-2000] [DSPE-PEG (2000)] biotin (Sigma-Aldrich, 880129P-10MG) to facilitate subsequent reactions with streptavidin-Cy3. Briefly, the DSPE-PEG (2000) biotin powder was dissolved in anhydrous ethanol to form a biotin storage solution (5 mg/ml). The biotin storage solution was then added to the concentrated KSHV virus to achieve a final concentration of biotin (0.1 mg/ml). After incubation at room temperature for 2 hours, the labeled KSHV and excess biotin were separated by illustra NAP Columns (Cytiva, 17085301) according to the manufacturer’s instructions. The labeled KSHV was stored at 4°C and used as soon as possible.
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