5x Passive Lysis Buffer
Cell lysis buffer to be used with the Renilla Luciferase Assay Kit or Firefly & Renilla Luciferase Single-tube Assay Kit.
5x Passive Lysis Buffer
PK-CA707-99912
30 ml
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5x Passive Lysis Buffer is a concentrated cell lysis buffer that is designed for use with the PromoKine Luciferase Reporter Assay Kit II (Renilla) or Luciferase Reporter Assay Kit III (Firefly&Renilla). These kits are supplied with sufficient 5x Passive Lysis Buffer to perform the stated number of assays with cells grown in 96 – 24 well plates. 5x Passive Lysis Buffer is sold separately for use with the kits when more lysis buffer (e.g. >100 µL/well) is required.
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Technical Library (10)
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How can I measure the ratio of successful transfections?
Transfection rates can directly be measured by (co-)transfecting a GFP-expressing plasmid. The percentage of cells that have taken up and expressed the foreign nucleic acid can be visualized with a fluorescence microscope (by green autofluorescence).
Another way of quantification is to transfect luciferase or beta-galactosidase expressing reporter vectors and to determine the activity of these enzymes as a measure of successful transfection. Several commercial cell based reporter assays are available for this purpose (Firefly and Renilla Luciferase Reporter Assay Kits; GFP reporter Assay Kit; beta-Galactosidase Activity Assay Kit; all available from PromoKine).Related Links and Documents
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What is the half-life of the Renilla and Firefly Assays?
PromoKine’ s Renilla and Firefly Assays (Luciferase Reporter Assay Kits I, II, and III) are flash luminescence assays. The approximate half-lives are 1-2 minutes for Renilla and 10 minutes for Firefly. The Luciferase Reporter HTS Kit has a half-life of about 3-5 hrs. However, the signal is about 1/10 of the flash luminescence assays.
Related Links and Documents
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When monitoring luciferase expression, can I add assay buffer to all my samples and read them simultaneously?
No. PromoKine’ s Renilla and Firefly Assay Kits (PK-CA707-30003; PK-CA707-30004; PK-CA707-30081) are based on flash luminescence. Samples should be treated and read individually. Alternatively, you may purchase the Firefly Luciferase Reporter HTS Kit (PK-CA707-30028). This Assay Kit is a proprietary mixture of substances that modify the enzymatic reaction to produce a long lasting signal (steady glow) by preventing the formation of adenyl-oxyluciferin at the enzyme surface.
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Can I use Promega’s Lysis Buffer or Assay Buffer with PromoKine Luciferase Reporter Assay Kits?
No. The buffers are based on different proprietary formulations.
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What’s the difference between Renilla and firefly luciferase?
Firefly luciferase is a 62 kDa protein isolated from beetles (Photinus pyralis), while Renilla luciferase is a 36 kDa protein from sea pansy (Renilla reniformis).
These enzymes differ in their substrate and cofactor requirements. Firefly luciferase uses luciferin in the presence of oxygen, ATP, and magnesium to produce light, while Renilla luciferase requires only coelenterazine and oxygen.
Firefly luciferase produces a greenish yellow light in the 550–570 nm range. Renilla luciferase produces a blue light of 480 nm.
These enzymes can be used in dual-reporter assays due to their differences in substrate requirements and light output.Related Links and Documents
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Why would I need to use the Dual Luciferase Reporter Assay Kit (Firefly & Renilla Single-tube Assay)?
Renilla is often used as an internal control for transfection efficiency while the Firefly luciferase vector is used for reporter activity. They also can be reversed, with Renilla as the reporter and Firefly as the internal control.
PromoKine’s Luciferase Reporter Assay Kit III (PK-CA707-30081) allows measurement of Firefly and Renilla luciferase activity in the same sample with high sensitivity and linearity.Related Links and Documents
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Why are my luminescence numbers different from those in the product information sheet?
Luminescence values are relative and depend on the experimental system. For example, luminometers from two different manufacturers each may have 6-log capability, but one may measure a range of 1 to 1,000,000 relative light units (RLU) while the other measures 0.01 to 10,000 RLU. Both instruments can measure a dynamic range of 6-log units, but they read out different arbitrary units.
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Which Luciferase Reporter Assay is more sensitive, Renilla or Firefly?
Assay sensitivity is dependent on many factors including the promoter strength of each vector, the transfection efficiency of each vector, and the cell type used. In general, Firefly luciferase is more widely used because it has a longer half-life and better stability than Renilla luciferase.
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I have a question regarding the Promokine Renilla Luciferase Reporter Assay (K-CA707-30004-1). Is the half life such that you recommend using a luminometer with injectors?
The half life of our Renilla luciferase substrate is approximately 2 min. Although it is possible to do readings manually, I would recommend a luminometer with injectors especially if you expect small differences between samples. This removes any variability from the manual readings as it is difficult to consistently add and read the samples in a timely fashion. However, we routinely do manual readings as well and it is possible to do them this way.
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Can I interchange the Renilla Lysis Buffer and Firefly Lysis Buffer?
The Passive Lysis Buffer (Renilla) is compatible with the Firefly Assay, but the Firefly Lysis Buffer is not compatible with the Renilla Assay.
Related Links and Documents
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How can I measure the ratio of successful transfections?
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Reference Literature (3)
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Alkyne-functionalized cationic polysiloxane polymers conjugated with targeting molecules by click reactions for DNA delivery
Yoshihiko et al.; Journal of Nanoscience and Nanotechnology. 2017 Jul;17(7):5081-5089(9)
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TOX3 plays a role in neuronal progenitor proliferation
Sahu et al.; Biochim Biophys Acta. 2016 Apr 11;1859(7):833-840
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Efficient generation of double heterologous promoter controlled oncolytic adenovirus vectors by a single homologous recombination step in Escherichia coli
Hoffmann and Wildner; BMC Biotechnol. 2006 Aug 3;6:36
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Alkyne-functionalized cationic polysiloxane polymers conjugated with targeting molecules by click reactions for DNA delivery
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