2-Hydrazinopyridine is an aromatic hydrazine reagent used to quantify the molar substitution ratio (MSR) of 4FB-modified proteins or other biomolecules. This aromatic hydrazine reacts with 4FB-modified biomolecules and forms a quantifiable hydrazone absorbance signature at 350 nm.
We DO think that our NanoLink™ streptavidin-coated beads might provide you with lower nonspecific protein binding to your biotinylated biomolecule (compared to competing products) for 2 reasons. (1) Because of the highly crosslinked streptavidin (using our unique, patented chemistry) there is less polymer surface area on the bead exposed for proteins or nucleic acids to adhere to. This in itself should reduce NSB for you. Our extensively crossliked streptavidin is the reason the biotin binding capacity of these beads is so high. (2) And…because of the high biotin binding capacity, you can use FEWER beads in your assay…again reducing nonspecific protein binding.
Once in solution, the peptide should either be used ASAP or frozen at –80 ºC. Be sure to aliquot the peptide solution into usable portions prior to freezing to avoid the need to freeze-thaw. TAT-HyNic peptide conjugation. A 3–5 mole excess of TAT-HyNic peptide should be used. Purification can be performed with a diafiltration column with a 5 kDa MWCO or by dialysis. Storage buffer should be PBS with 0.5% azide.
Our recommended storage time (shelf life) is 18 months. After that time, you can reconstitute one of your vials, run on an HPLC, and see if you observe a single peak. If yes, then it’s still OK. If multiple peaks are observed, then some degradation may have occurred.
These reagents are stable for up to 1 month if stored at 4oC.
4FB-modified proteins are stable at 4oC for up to one year. HyNic-modified proteins are not stable and should be used the same day they are modified, preferably. HyNic-modified proteins can be stored at –80?C in small, single-use aliquots, in conjugation buffer. No need to add glycerol. And, do not re-freeze after used – reason for the aliquots. Should be good for several months. You want the proteins frozen, not moving around in solution, with possible HyNic-amino group interaction.
If you would like recommendations, we welcome you to contact technical support (email@example.com).
All of the beads should be attached to antibody if you use a 10 to 20-fold molar excess of biotinylated product. Washing away unbound antibody is straightforward – you can magnetize the beads and wash them to remove unbound antibody. You can repeat this a few times, it is not trivial to remove any unbound beads from bound beads.
Random DNA sequences may bind to the surface of the magnetic beads, but only very weakly, if at all. The non-biotinylated sequences can easily be washed away from the beads. The crosslinked streptavidin doesn’t prevent binding.
We have 2 sizes of beads to fit the needs of specific applications. Some customers need a uniform sized bead. The MagnaLink™ beads offer this. For MOST applications however, this is not necessary. Some microarray applications might require a certain size as well. The larger bead will settle faster if that might be an issue in your application.
Higher binding capacity means greater capture using less bead mass. Less bead mass for your immobilization assays results in better assay capture and increased savings.
We currently offer two different types of magnetic beads; NanoLink™ is a less-uniform bead mixture which has 150 and 800 nm diameter beads, and MagnaLink™ is a very uniform bead with a 2.8 µm diameter. Both bead types are made with magnetite encapsulated by a non-styrene polymer surface. Unless you are particularly concerned with uniformity (e.g., for NMR, flow cytometry, etc.) the NanoLink™ beads are particularly suited for high-capacity capture of oligos, peptides, and antibodies. Both bead types come in three different varieties; streptavidin coated, 4FB linker coated, and amine coated. As a custom service, we can get other bead types for you, and link them to 4FB or streptavidin. And, we can add a linker with an extended spacer, but currently offer only streptavidin-coated, 4FB-coated, and amino beads as catalog products.
Bead size: bimodal (non-uniform) distribution (~150 nm to ~800 nm)
Density : 1.5 g/cm3
Magnetite content (Fe3O4): 40% (w/w)
Magnetic moment: 25 emu/g (electromagnetic units per gram)
Bead size; 2.8 micron (uniform distribution)
Density : 1.9 g/cm3
Magnetite content (Fe3O4): 50% (w/w)
Magnetic moment: N/A
The CleanAmp™ primers were created to reduce primer dimer and mis-priming however the CleanAmp group for the primers is situated on the internucleotide phosphate linkages.
This makes them very similar to a phosphorothioate or methyl phosphonate oligo which allows for nuclease resistance. I would recommend the precision primers since there are two CleanAmp groups for added nuclease stability. The CleanAmp primers 3’ OH group is exposed (not protected). The protecting group for the primers is different than for the CleanAmp dNTPs which is is blocking the 3’ OH.