A Rapid And An Efficient Method To Remove Unreacted Small-Molecules Using A Versatile Resin Packed In A Convenient Spin-And-Go Format

A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format. Removal of unreacted or residual compounds in the sample-preparation workflow is a key success factor for downstream analysis and processing of biomolecules. For example, during sample preparation of biomolecules such as proteins or nucleic acids, there is often a need to label the biomolecules with dyes, affinity tags, radioactive labels, and mass tags. In other instances, there is a need to chemically modify biomolecules such as to reduce, oxidize, methylate, etc. During these treatments, some amount of the labeling or chemical agent remains in the sample as an unreacted label/chemical. These unreacted small molecules can cause several issues during downstream analysis or use of the biomolecule. For example, free unreacted fluorescent dyes after bioconjugation of protein cause non-specific binding of free-dye resulting in high background issues during fluorescent imaging. Commonly used clean-up methods following sample preparation, such as dialysis, are tedious and lengthy with multiple buffer exchanges sometime resulting in loss of protein due to aggregation. Alternatively, size-exclusion chromatography requires expensive instrumentation in addition to lengthy set up time. We have developed the Thermo Scientific™ PierceTM Dye and Biotin Removal Spin Columns, a novel resin that is packed in an easy to use “Spin and Go” format of varying spin column sizes (0.5 mL, 2mL, 5 mL and 10 mL) to accommodate a range of sample volumes (100 uL- 4 mLs). The resin is highly specialized to produce exceptional protein recovery and can be used effectively to remove 4 different class of small molecules: non-conjugated fluorescent dyes, biotinylation reagents, reducing agents, and crosslinkers. It is a multimodal resin with size exclusion properties and proprietary surface chemistry that allows removal of the above-mentioned small molecules, while the size exclusion property allows for the biomolecule to be efficiently recovered. Our results indicate efficient dye removal and protein recovery from fluorescent conjugated antibodies. The results also indicate removal of reducing agents such as TCEP at a concentration as high as 25 mM from protein samples, while recovering \u003e90% of the added protein. Results also indicate \u003e90% removal of biotinylated molecules and crosslinkers from Protein samples with \u003e90% protein recovery. Our results show that the efficiency and binding capacity for removal of small molecules using this resin is better than any resin in the market. This is exemplified by immunofluorescent analysis of ZO-1 monoclonal antibody conjugated Alexa Fluor™ 488 which was purified using PierceTM Dye and Biotin Removal Spin columns and shows high intensity and lesser background in Caco-2 cells than the non-purified conjugate. Citation Format: Deven Kent Etnyre, Ramesh Ganapathy, Marie Nlend, Chris Etienne, Atul M. Deshpande. A rapid and an efficient method to remove unreacted small-molecules using a versatile resin packed in a convenient Spin-and-Go format [abstract]. In: Proceedings of the Annual Meeting of the American Association for Cancer Research 2020; 2020 Apr 27-28 and Jun 22-24. Philadelphia (PA): AACR; Cancer Res 2020;80(16 Suppl):Abstract nr 2851.