Full‐length SARS‐CoV‐2 Spike (2P‐stabilized, C‐terminal Histidine/Avi‐tagged) was obtained from BEI resources (Manassas, VA, Cat. NR53524) and biotinylated using a BirA ubiquitin ligase (Avidity, Aurora, CO, Cat. Bir500A) following the manufacturer's recommended protocol. Biotinylated spike protein was purified using a 40 K molecular weight cut‐off (MWCO) 2 ml Pierce Zeba™ desalting column (Thermo Fisher Scientific, Waltham, MA, Cat. 87768), and mixed with streptavidin BV421 (BD, Cat. 563259) or streptavidin allophycocyanin (APC) (BD Cat. 554067) separately at a 20:1 ratio (~6:1 molar ratio) as previously described [3 (link)]. Tetramerized Spike probes were stored at 4°C until use. Just prior to probe staining, spike protein probes were added one‐by‐one to FACS wash buffer (1x PBS, 2% fetal bovine serum) containing 5 μM free d‐biotin (Avidity, Cat. Bir500A). Both streptavidin‐fluor conjugates were used to stain dimethyl sulfoxide (DMSO) control samples to further verify the absence of significant frequencies of nonspecific streptavidin‐binding B cells.
Pierce zeba desalting column
Manufactured by Thermo Fisher Scientific
The Pierce Zeba™ desalting column is a size-exclusion chromatography device designed to remove small molecules, such as salts, from protein-containing samples. It functions by allowing the passage of small molecules through a porous matrix while retaining larger proteins. This straightforward process facilitates the buffer exchange or desalting of protein samples, preparing them for further analysis or applications.
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Lab products found in correlation
Streptavidin bv421, by BD (1 mentions)
Streptavidin allophycocyanin apc, by BD (1 mentions)
D biotin, by Avidity (1 mentions)
Hispur ni nta resin, by Thermo Fisher Scientific (1 mentions)
Pei reagent, by Thermo Fisher Scientific (1 mentions)
Recombinant human n slit2, by Thermo Fisher Scientific (1 mentions)
Toxinsensor chromogenic lal endotoxin assay kit, by GenScript (1 mentions)
2 protocols using pierce zeba desalting column
1
SARS-CoV-2 Spike Protein Biotinylation
2
Large-scale Production of Bio-inactive N-SLIT2ΔD2
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Large-scale production of bio-inactive human N-SLIT2ΔD2 was performed by transfecting FreeStyle 293-F cells with human N-SLIT2ΔD2 cDNA (1 ug/mL) using PEI reagent (Polyethyleneimine, linear, M.W. 25,000, Thermo Fisher Scientific) (Patel et al., 2012 (link)). After 5 days, the culture medium was loaded onto HisPur Ni-NTA resin (Thermo Fisher Scientific), washed with imidazole (35 mM), eluted with imidazole (250 mM), and desalted using a Pierce Zeba desalting column (7 K MWCO; Thermo Fisher Scientific). Protein molecular weight was determined by immunoblotting using anti-6XHIS-HRP conjugated antibody. Protein activity was assayed by using a spreading assay in RAW264.7 cells, as described previously (Bhosle et al., 2020 (link)). Recombinant human N-SLIT2 was purchased from PeproTech (Cranbury, NJ, USA). Endotoxin levels in all preparations were measured using ToxinSensor Chromogenic LAL Endotoxin Assay Kit (GenScript, Piscataway, NJ, USA) and were less than 0.05 EU/ml.
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