OVA was conjugated in 3-fold molar excess to mAbs via thioether linkages as described (37 ). Unconjugated OVA was removed from mAb-OVA conjugates using 100 kDa cut-off spin columns (Millipore). Retained mAb-OVA conjugates were resuspended in PBS, treated with polymyxin B (Sigma) overnight at 4C to remove endotoxin, sterile filtered (0.2 μM) and stored at −20C until use. ELISA assays (described below) were used to confirm Ag-mAb conjugation and determine the final concentration of OVA and mAb. The quantities of OVA per mg of mAb were as follows: OVA-DEC205, 0.86 mg; OVA-G28-1, 0.85 mg; OVA-UW80.1, 0.84 mg; and OVA-Siglec-H, 0.55 mg.
100 kda cut off spin columns
Manufactured by Merck Group
Sourced in Morocco
100 kDa cut-off spin columns are a type of laboratory equipment used for the separation and purification of macromolecules, such as proteins, from complex mixtures. These columns contain a semi-permeable membrane that allows the passage of molecules with a molecular weight less than 100 kDa, while retaining larger molecules. The core function of these spin columns is to facilitate the efficient separation and concentration of the desired macromolecules from the sample.
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Lab products found in correlation
2 protocols using 100 kda cut off spin columns
1
Antigen-Antibody Conjugation and Endotoxin Removal
2
Melanocyte-Derived Extracellular Vesicle Isolation
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For EV isolation, confluent melanocytes were washed with PBS and grown in M-254 medium for 48 hours. Conditioned medium was collected and centrifuged at 500 Â g for 10 minutes, then at 3,000 Â g for 20 minutes, and finally at 100,000 Â g for 2 hours. The resulting EV-containing pellet was resuspended in PBS and stored at À80 C. Alternatively, conditioned medium was clarified by filtration through 0.45-mm pore filters (Millipore, Billerica, MA) and concentrated using 100-kDa cutoff spin columns (Millipore). EVs were isolated using an ExoQuick-TC kit (SBI, San Jose, CA) according to the manufacturer's instructions. A volume of 20 ml of isolated EVs was analyzed by western blot and silver staining.
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