Secrets of Unit Definitions & Assays

Secrets of Enzyme Units: Definitions & Assays

  
Enzyme units vary and the differences can make comparing products from different sources difficult.  We’ve looked into this because we occasionally hear that brand X is much cheaper that our AMV RT.Well, appearances can be deceiving. Although unit definitions may appear identical, assay conditions do vary and one can’t assume that the unit potency is the same between suppliers.

In order to prove this point, Life Sciences Advanced Technologies (LSAT) purchased AMV RT form several different sources and tested them in our laboratories using a common assay procedure. We found a lot of variation. Here are the results with supplier units express as a percentage of LSAT’s unit.

  
Supplier Claimed U/μL Assayed Value U/μL Supplier vs. LSAT Unit Suppliers Unit Definition  
LSAT 14.6 14.1 97% 1 Unit = amount required to   incorporate 1 nmol of TTP into acid-insoluble material in 10 min. at 37ºC
Gene Choice 10 5.7 57% 1 Unit = amount required to   incorporate 1 nmol dTTP into acid precipitable material in 10 min. at 37ºC
Invitrogen (cloned) 15 13 87% 1 Unit = amount required to incorporate 1 nmol dTTP into acid-precipitable material in 10 min. at 37ºC, using polyA:oligo (dT)25 as a primer
NE Biolabs 25 4.1 23% 1 Unit = amount required to  incorporate 1 nmol dTMP into acid-insoluble form in 10 min. at 37ºC using Poly A:  oligo (dT) as template primer
Promega 24 4.6 19% 1 Unit = amount required to incorporate 1 nmol deoxynucleotide into acid-precipitable material in 10 min. at 37ºC
Seikagaku 26 9.7 38% 1 Unit = amount required to  incorporate 1 nmol dTMP into acid-insoluble material in 10 min. at 37ºC
USB (Affymetrix) 15 3 20% 1 Unit = amount required to incorporate 1 nmol TMP into acid-insoluble material in 10 min. at 37ºC
 
Claimed U/µL: the unit concentration from the supplier’s product labeling.Assayed Value U/µL: the average of 2 assays (4 for LSAT).

Supplier vs. LSAT Unit: expressed as the percentage deviation form the value claimed by the supplier.

LSAT Assay conditions: 4.0mM DTT, 50mM Tris-HCI (pH 8.3), 40mM KCI, 6mM MgCI2, 0.4mM r(A)n:(dT)12.18, 0.5mM (3H)-TTP, AMV RT diluted with 0.O1M phosphate buffer (pH 7.2)

  
Conclusion:Life Sciences Advanced Technologies maintains the closest ratio between claimed units and assay value.

If you use significant amounts of an enzyme, it is worth your time to assay different suppliers’ products to ensure a meaningful comparison and good value.

  
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Trehalose Review Articles

Trehalose Review Articles
We use trehalose to stabilize some of our enzymes during a freeze-drying process. We used to  purchase trehalose dihydrate from a well-known St. Louis company. However, quality was not consistent, forcing us to screen lots for the presence RNase which can compromise our products. Sometimes there was not lot available that would meet our criteria.

Now, we process our own trehalose, both powder and sterile solution for internal use and for sale.

Stabilization, Freeze-Drying, etc.
Enzyme and antibody stabilization is our main interest. Other applications for cells and tissues are beyond our experience, but for those who are interested here is a short list of review articles.
  1. Chen T et al.: (2000) Literature review: supplemented phase diagram of the trehalose-water binary mixture. Cryobiology  v40 pp277-282.
  2. Chen, Y and Z G Lu (2006) Research progress on trehalose used in lyophilization of blood cells – review.   Zhongguo Shi Yan Xue Ye Xue Za Zhi v.14 pp 416-418.
  3. Crowe, J H et al (2003) Stabilization of membranes in human platelets freeze-dried with trehalose., Chemistry and Physics of Lipids. V.122, no.1-2 pp 41-52.
  4. Elbein, A D et al. (2003) Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies.  Glycobiology v. 13 no. 4 pp. 17R-27R
  5. Elbein, A D et al. (2003) Review: New insights on trehalose: a multifunctional molecule. Glycobiology, v13 (4j) 17R-27R
  6. 7.    Franzetti , A et al. (2010) Production and Applications of Trehalose Lipid Biosurfactants. European Journal of Lipid Science and Technology. v.9999 no. 999A
  7. Iturriaga, G et al. (2009) Trehalose Metabolism: From Osmoprotection to Signaling. International Journal of Moeculr Sciences v.10 no.9, pp 3793-3810;
  8. Jain N K and I Roy (2009) Effect of trehalose on protein structure. Protein Science v.18 pp 24-36
  9. Richards, A B et al. (2002) Trehalose: a review of properties, history of use and human tolerance, and results of multiple safety studies. Food and Chemical Toxicology  v.40 no.7 pp.871-98.
  10. Ryll, R et al. (2001) Immunological properties of trehalose dimycolate (cord factor) and other mycolic acid-containing glycolipids–a review. Microbiology and Immunology v.45
  11. Schwegman, J J et al. (2005) Practical Formulation and Process Development of Freeze-Dried Products. Pharmaceutical Development and Technology v10 (2)151 – 173 A1
  12. Teramoto , N et al. (2008) Trehalose and Trehalose-based Polymers for Environmentally Benign, Biocompatible and Bioactive Materials. Molecules v.13, no.8, 1773-1816
  13. Ragoonanan, V and A Aksan (2007) Protein Stabilization. Transfusion Medicine and Hemotherapy v.34 pp 246–252

Molecular Biology Grade Trehalose (Yep, this is the product plug)
Our trehalose dihydrate tests negative for RNase/DNase and endotoxin, plus its very low in other contaminants. Trehalose Solution is a 1M solution made from our Molecular Biology Grade and sterile filtered in a validated process. Full specifications and pricing may be found by following the links above.
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Secrets of Unit Definitions & Assays

Secrets of Enzyme Units: Definitions & Assays   Enzyme units vary and the differences can make comparing products from different sources difficult.  We’ve looked into this because we occasionally hear that brand X is much cheaper that our AMV RT.Well, appearances can be deceiving. Although unit definitions may appear identical, assay conditions do vary and [...]

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Trehalose Review Articles

Trehalose Review Articles We use trehalose to stabilize some of our enzymes during a freeze-drying process. We used to  purchase trehalose ...

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