Monovalent MTRIPs (mMTRIPS) consist of a NeutrAvidin core bound to an mRNA-targeting ligand via an aromatic hydrazine and aldehyde linkage (Hynic-4FB, Solulink) and to four biotinlated and fluorescently labeled reporter oligonucleotides. The mRNA-targeting ligand – containing a 5′ thiol modification (Biosearch Technologies) – are first conjugated to a 30x molar excess of maleimide HyNiC crosslinker (MHPH, Solulink). NeutrAvidin (Sigma) is labeled with 4FB groups (S-4FB, Solulink) to achieve a ratio of 2 4FB groups per molecule, as quantified by colorimetric reaction with 2-hydrazinopyridine (Solulink) by UV-Vis (Abs=350). The oligonucleotides and NeutrAvidin are separately filtered and buffer exchanged then combined according to the manufacturer protocol. The concentration of the resulting modified NeutrAvidin is quantified using a BCA Protein Assay Kit (Pierce). The reporter oligonucleotides contain a 5'-biotin modification and one dT-C6-NH2 modification for conjugation with Cy3B-NHS ester (GE Healthcare) using manufacturer protocols. Free dye was removed using 3kD Nanosep spin columns (Pall Corp.). The purified ligands were resuspended in 1xPBS and mixed at a 5:1 molar ratio with the modified NeutrAvidin for 1 hour at RT. Free ligands were removed using 30 kD Nanosep spin columns.
3kd nanosep spin columns
Manufactured by Pall Corporation
The 3kD Nanosep spin columns are a product from Pall Corporation designed for sample preparation and purification. These centrifugal devices are used to separate and concentrate macromolecules, such as proteins and peptides, based on their molecular weight. The columns feature a 3kD molecular weight cutoff membrane that allows the separation of molecules below this threshold, making them suitable for various laboratory applications that require sample concentration and desalting.
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Lab products found in correlation
2 protocols using 3kd nanosep spin columns
1
Monovalent mRNA-Targeting Reporter Probes
2
Monovalent mRNA-Targeting Reporter Probes
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Monovalent MTRIPs (mMTRIPS) consist of a NeutrAvidin core bound to an mRNA-targeting ligand via an aromatic hydrazine and aldehyde linkage (Hynic-4FB, Solulink) and to four biotinlated and fluorescently labeled reporter oligonucleotides. The mRNA-targeting ligand – containing a 5′ thiol modification (Biosearch Technologies) – are first conjugated to a 30x molar excess of maleimide HyNiC crosslinker (MHPH, Solulink). NeutrAvidin (Sigma) is labeled with 4FB groups (S-4FB, Solulink) to achieve a ratio of 2 4FB groups per molecule, as quantified by colorimetric reaction with 2-hydrazinopyridine (Solulink) by UV-Vis (Abs=350). The oligonucleotides and NeutrAvidin are separately filtered and buffer exchanged then combined according to the manufacturer protocol. The concentration of the resulting modified NeutrAvidin is quantified using a BCA Protein Assay Kit (Pierce). The reporter oligonucleotides contain a 5'-biotin modification and one dT-C6-NH2 modification for conjugation with Cy3B-NHS ester (GE Healthcare) using manufacturer protocols. Free dye was removed using 3kD Nanosep spin columns (Pall Corp.). The purified ligands were resuspended in 1xPBS and mixed at a 5:1 molar ratio with the modified NeutrAvidin for 1 hour at RT. Free ligands were removed using 30 kD Nanosep spin columns.
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