2-Sulfobenzaldehyde is a water-soluble aromatic aldehyde reagent used to quantify the molar substitution ratio (MSR) of HyNic-modified proteins or other biomolecules. This aromatic aldehyde reacts with HyNic-modified biomolecules and forms a quantifiable hydrazone absorbance signature at 390 nm.
Author Archives: TriLink
How does one determine how many 4FB molecules are bound to my biomolecule?
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.
Will NanoLink™ streptavidin beads experience less nonspecific binding (NSB) of biotinylated nucleic acids or biotinylated proteins than other beads?
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.
If my TAT-HyNic peptide is dissolved in conjugation buffer; should I store at –20ºC? How long the does the solution stay active? What molar excess of TAT-HyNic should I use for antibody conjugation? After conjugation, can purification be done by using a desalting column or by dialysis? What kind of buffer is best for antibody-TAT storage?
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.
What is the stability of S-HyNic (if stored at –20ºC, dessicated)?
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.
How stable are HyNic and 4FB reagents (stored in DMF from TriLink)?
These reagents are stable for up to 1 month if stored at 4oC.
How stable are HyNic and 4FB-modified proteins (stored in aqueous solution)?
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.
How do I know which linker to put on each of my 2 biomolecules?
If you would like recommendations, we welcome you to contact technical support (sales@trilinkbiotech.com).
How do I ensure that all of my beads become labeled with biotinylated biomolecules?
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.
Are the primers mainly reducing primer-dimer or they are also protected from exonuclease activity of proof-reading polymerase? In other words, is the 3’OH of the primer exposed or available to low temperature before they are heat inactivated.
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.