MarkerGene™ Long Wavelength Fluorescent Lipase Assay Kit

Product ID: M1214



Unit SizePriceQuantity 
1kit
$280.19
  • Buy 5 for $224.16 each and save 20%

Availability: In stock


Description

This kit allows fast and easy measurement of lipase activity in vitro, in cell preparations or in vivo using the long wavelength substrate resorufin oleate.

Lipases are a family of enzymes that release fatty acids from triacylglycerols in a site specific manner. Most lipases have optimum activity for the primary ester groups of triglycerides, while some lipases remove fatty acyl groups from either the C-1 or C-3 acyl positions. The substrate is typically not a single molecule, but a nonaqueous phase of aggregated lipid. Lipase activity, ubiquitous among most cells, can be monitored using our long wavelength fluorescent substrate, resorufin oleate(M1208) contained in the kit. Upon cleavage, the fluorescent compound, resorufin (M0202) is released and activity measurements are easily obtained either in vitro, in cell preparations, or in vivo. The kit contains enough substrate for numerous assays and control experiments, and also contains reference standards and a detailed protocol for use. See the references below for more information and applications.

Technical Data
SKU M1214
Unit Size 1kit
Detection Method Fluorescence

References and Citations

Citations:

  • Henkel M, Schmidberger A, Vogelbacher M, Kühnert C, Beuker J, Bernard T, Schwartz T, Syldatk C, Hausmann R. (2014) "Kinetic modeling of rhamnolipid production by Pseudomonas aeruginosa PAO1 including cell density-dependent regulation." Appl Microbiol Biotechnol. 98(16):7013-25.
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References:

  • Main LA, Okumura-Noji K, Ohnish, T, Yokoyama S. (1998) "Cholesteryl ester transfer protein reaction between plasma lipoproteins." J. Biochem. (Tokyo) 124: 237-243.
  • Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O. (1994) "Bacterial Lipases" FEMS Microbiol. Rev. 15(1): 29-63.
  • Dousset N, Negre A, Salvayre R, Rogalle P, Dang QQ, Douste-Blazy L. (1988) "Use of a fluorescent radiolabeled triacylglycerol as a substrate for lipoprotein lipase and hepatic triglyceride lipase." Lipids 23: 605-608.
  • Galla HJ, Theilen U, Hartmann W. (1979) "Transversal Mobility in Bilayer Membrane Vesicles: Use of Pyrene Lecithin as Optical Probe" Chem Phys Lipids 23: 239-251.
  • Dergunov AD, Shuvaev VV, Perova NV. (1989) "Topo-dynamic characteristics of human plasma VLDL apolipoproteins and efficiency of triacylglycerol hydrolysis by lipoprotein lipase." Biochim Biophys Acta 1005(1): 79-86.
  • Liodakis A, Drew J, Chan RY, Sawyer WH. (1991) "Spectrofluorometric determination of lipase activity." Biochem Int 23(5): 825-34.
  • Negre A, Salvayre R, Dousset N, Rogalle P, Dang QQ, Douste-Blazy L. (1988) "Hydrolysis of fluorescent pyrenetriacylglycerols by lipases from human stomach and gastric juice." Biochim Biophys Acta 963: 340-348.
  • Hendrickson HS. (1994) "Fluorescence-bases assays of lipases, phospholipases, and other lipolytic enzymes." Analyt Biochem 219: 1-8.
  • Negre-Salvayre A, Abouakil N, Lombardo D, Salvayre R. (1990) "Hydrolysis of fluorescent pyrene-acyl esters by human pancreatic carboxylic ester hydrolase and bile salt-stimulated lipase." Lipids 25(8): 428-434.
  • Negre A, Maret A, Douste-Blazy L, Gatt S, Salvayre R. (1988) "Relative fluorescence of normal and acid lipase-deficient cultured fibroblasts following administration of pyrene decanoic acid." Biochim Biophys Acta 960: 401-409.
  • Negre A, Salvayre R, Dagan A, Gatt S. (1985) "New fluorometric assay of lysosomal acid lipase and its application to the diagnosis of Wolman and cholesteryl ester storage diseases." Clin Chim Acta 149: 81-88.
  • Rosseneu M, Taveirne M, Caster H, Van Biervliet J. (1985) "Hydrolysis of very-low-density lipoproteins labeled with a fluorescent triacylglycerol: 1,3-dioleoyl-2-(4-pyrenylbutanoyl)glycerol." Eur J Biochem 152: 195-198.
  • Mantulin WM, Massey JB, Gotto AM, Pownall HJ. (1981) "Reassembled Model Lipoproteins." J Biol Chem 256(21): 10815-10819.
  • Liodakis A, Drew J, Chan R, Sawyer WH. (1991) "Spectrofluorometric determination of lipase activity." Biochem Intern 23(5): 825-834.
  • Arpigny J L, Jaeger KE.(1999) "Bacterial lipolytic enzymes: classification and properties." Biochem J 343: 177-183.
  • Kouker G, Jaeger KE. (1987) "Specific and sensitive plate assay for bacterial lipases." Appl Environ Microbiol 53(1): 211-3.
  • Howard GT, Vicknair J, MacKie RI,. (2001) "Sensitive plate assay for screening and detection of bacterial polyurethanase activity." Lett Appl Microbiol 32(3): 211-4.
  • Salvayre R, Negre A, Radom J, Douste-Blazy L. (1986) "Fluorometric Assay for Pancreatic Lipase" Clin Chem 32(8): 1532-1536.
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Technical Support

Question about this product? Ask a Scientist!

We pride ourselves on the high quality of our products and want you to get the best possible results from your assays. If you have any questions about this product or need help optimizing your protocol check out the product FAQs below or ask your own question and one of our expert scientists will get back to you asap:


⚛ Question: I was interested in running the lipase assay (M0612) at varying temperatures (10C-40C) but was worried about the level of non-specific hydrolysis as well as the function of the resorufin oleate at higher temperatures. Is there any way to reduce the non-specific hydrolysis at higher temperatures or will the assay even function at 37C and 40C??
➸ Answer: I believe the assay should work at the temperatures you have specified, though I have not actually tried it so I cannot be certain. I have found the best way to reduce non-specific hydrolysis of the substrate is to cool the reagents on ice before mixing.
⚛ Question: I had a question regarding the calculation of activity, specifically with Step 9 in the protocol. To generate the calibration curve, do you take the rate (RFU v time) of each standard (if so, do you generate a log-log plot to get a linear slope?) or the final normalized fluorescent reading at 20min of each standard to generate a std curve?
➸ Answer: The fluorescence readings will only be able to give you "relative" lipase concentrations. You would need to purify the enzyme to actually get a quantitative value of the lipase activity or kinetics. If you have a purified enzyme, you can run a standard calibration curve as you describe.
But be aware that the tissues may have multiple lipase enzymes present. The different isoforms of the lipases could have different Vmax values. And the crude cell lysate may also mask the lipase activity as well. Using the kit with cell lysates will therefore only tell you that the lipase activity in cell line 1 is say 50% of that in cell line 2, without doing some other purification. You can do this by either plotting the kinetics, as you mention, or measuring at a single time point for all the samples, i.e. 20 min. or 60 min.
The resorufin standard is used to convert the RFU values from the plate reader or fluorometer into actual concentrations of substrate turnover. You don't really need a standard curve for this, you can just use one point, but it is good to see what the linear range is for your instrument. If your enzyme assay at a certain time period gives a RFU reading of, say 5000, then you can use the curve to calculate the concentration of resorufin that gives the same RFU signal. This will then correlate to the concentration of resorufin generated in the assay and (by definition) the concentration of substrate that has been used by the enzyme at that point. This allows you to calculate velocities (umole/min/mL) from the RFU data.
⚛ Question: Is there a specific plate that is recommended for this assay?
➸ Answer: We recommend the use of flat, clear bottomed 96 well plates for this kit. It may be additionally useful to use plates that are black on the sides to prevent crosstalk between wells. For example Corning product 3603.
⚛ Question: Do you know if this kit can be used to assay lipase activity of bacteria ?
➸ Answer: Unfortunately, we have not tested the assay with bacteria. I would point you to the following references where this type or assay and reagent has been used in bacteria.
  • Arpigny JL, Jaeger KE (1999)"Bacterial lipolytic enzymes: classification and properties." Biochem. J. 343: 177-183.
  • Kouker G, Jaeger KE (1987) "Specific and sensitive plate assay for bacterial lipases." Appl Environ Microbiol 53(1): 211-3.
  • Howard GT, Vicknair J, MacKie RI (2001) "Sensitive plate assay for screening and detection of bacterial polyurethanase activity." Lett. Appl. Microbiol. 32(3): 211-4.
  • Jaeger KE, Ransac S, Dijkstra BW, Colson C, van Heuvel M, Misset O (1994) "Bacterial lipases." FEMS Microbiol Rev. 15(1):29-63.