Luciferase Cell Lysis Buffer

产品编号：B3321S 品牌：New England Biolabs 原厂货号：B3321S
产品分类：蛋白质表达与纯化 > 蛋白质样本制备 > 蛋白提取
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包装：

运保温度:

4°C

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描述：

Description:
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.

Reagents Supplied:
5X Luciferase Cell Lysis Buffer

1X Luciferase Cell Lysis Buffer:
1 X concentration obtained by dilution of 5X buffer with distilled water

Protocol

Dilute LCLB (5X) with dH2O to 1X concentration.
Aspirate the growth media from wells.
Wash the cells once with PBS (pH 7.4) and aspirate.

Add the appropriate volume of 1X LCLB to each well (See Table below):

Culture Vessel	Surface (cm2)	Volume of 1X LCLB
98 well	0.32	25 µl
24 well	0.95	75 µl
12 well	1.9	150 µl
35 mm dish	3.8	250 µl
6 well	9.5	800 µl
60 mm dish	21	1.5 ml
100 mm dish	55	2.5 ml

Incubate at room temp for 15-20 min on an orbital shaker (making sure the surface in a well is completely covered with the buffer).
Use 5-20 µl of cell lysate for assaying.

注意事项：

Usage notes:

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 . 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 .
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.

Usage notes:

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.