Luciferase Cell Lysis Buffer (LCLB) is a proprietary formulation developed to produce mammalian cell lysates for reporter assays. This lysis buffer is compatible with reagents for assaying the activity of Gaussia as well as other luciferases (e.g. Renilla & Firefly) and β-galactosidase (Figure 1A-D).
Figure 1: LCLB compatibility with commonly used reporter assay systems NIH3T3 cells were transfected either with (A) pCMV-GLuc Control Plasmid (NEB #N8081S), (B) a lacZ-expressing vector, (C) a Renilla-expressing vector or (D) a Firefly-expressing vector, using TransPass D2 Transfection Reagent (NEB #M2554S). The supernatants from CMV-GLuc-transfected cells were saved for the assay at 24 h after transfection. All transfected cells were then washed once with PBS (pH 7.4) and lysed with either Luciferase Cell Lysis Buffer or commercially available lysis buffers recommended for each reporter system, respectively. Cell lysates (20 µL out of 100 µL total lysate per sample) were analyzed for GLuc or Renilla luciferase activity using the GLuc Assay Kit (NEB #E3300S/L), β-galactosidase activity using Galacto-Light System (ABI), and Firefly luciferase activity using Luciferase Assay System (Promega). Gaussia luciferase activity was also assayed from the supernatants (20 µL out of 500 µL growth medium of CMV-GLuc-transfected cells per sample).
Depending on the cell type, some cells may need larger volume and longer incubation time for sufficient lysis. One can determine whether or not the lysis is sufficient by assaying the activity of intracellular Gaussia luciferase using the pCMV-GLuc Control Plasmid (NEB #N8081). Although the Gaussia luciferase produced from pCMV-GLuc is secreted into the culture medium, at a given time point, 5-10% of the total activity should reside in the lysate (Figure 1A).
Normally, there is no need to remove the cell debris by centrifugation before assaying Gaussia luciferase activity. If additional assays are to be performed (e.g. protein concentration, SDS-PAGE, etc.), it is best to centrifuge the cell lysate to remove cell debris.
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The Product Summary Sheet, or Data Card, includes details for how to use the product, as well as details of its formulation and quality controls. The following file naming structure is used to name the majority of these document files: [Catalog Number]Datasheet-Lot[Lot Number]. For those product lots not listed below, please contact NEB at [email protected] or fill out the Technical Support Form for appropriate document.)
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