POLARIC®-NHS

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  POLARIC®-NHS
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  Properties of POLARIC-NHS

  Product name	target	reaction	Absmax (nm)*	FLmax (nm)*
  POLARIC-NHS	solvent polarity	reversible	480	592

  *Fluorescence wavelength changes upon the solvent polarity.

FAQ

• Q In what situation does POLARIC change the color?
  A

  It is reported that the fluorescent wavelength of POLARIC is changed by the composition of lipid (Chem. Lett. 2011, 4, 989-991). On the other hand, POLARIC show no significant difference of fluorescence wavelength by pH changes.

• Q Tell me the efficient labeling method using NHS reagent.
  A

  Prepare purified antibody (or other proteins). If you intended to crude protein sample, we recommend to purify protein using either affinity column, ultrafilteration or gel filteration before the labeling to increase labeling efficiency. In addition, avoid to use Tris-buffer, because Tris has primary amine and it strongly inhibit the reaction of NHS to the target protein.

  In some cases, reaction at 4^oC for overnight gives higher degree of labeling compared with reaction at 37^oC for 1 hour.

• Q How can I get molar extinction coefficient of a protein at 280 nm?
  A

  Molar extinction coefficient of protein at 280 nm can be calculated by the methods of Gill and von Hippel (1989) Analytical Biochemistry, 182: 319-326  and Anthis and Clore (2013) Protein Science 22:851-858.

  You may find Web services to obtain them in the following sites. Goryo Chemical do not support the usage of these sites and use them according to the Terms and Conditions in each sites.

  http://protcalc.sourceforge.net/
  http://web.expasy.org/protparam/
  http://nickanthis.com/tools/a205.html

• Q How can I prepare 0.1 M sodium bicarbonate buffer? Why this buffer is required?
  A

  Dissolve sodium bicarbonate (NaHCO₃) to pure water and adjust the concentration to be 0.1 M. The pH should be in the range of 8.0 to 8.4. Alternatively, you can adjust pH by adding a small amount of Na₂CO₃  or  HCl.

  The reason to recommend this buffer is that the reaction of NHS ester and primary amines is more efficient at alkaline pH. Instead, you can use other alkaline buffers such as HEPES/phosphate/borate buffers.  Avoid to use Tris buffer because it has primary amines and inhibit the reaction of NHS with the target molecules.

• Q My question is not in this FAQ list.....
  A

  Please also refer the frequently asked questions on fluorescent probes

   

Reference