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Beta-Galactosidase Colorimetric Assay
50 Tests in 35mm plate
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| Catalog Number: 8068 |
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Introduction
Beta-Galactosidase, an important reporter gene marker encoded by
lacZ, is commonly used for monitoring transfection efficiency in
cultured cells and identifying expression of recombinant fusion
genes. The ScienCell™ beta-galactosidase Colorimetric Assay
provides an easy-to-use method to detect beta-galactosidase by staining
cells with 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-gal)
at pH 7.4.
Kit Components
| Cat.
No. |
Vials
|
Reagents |
Quantity
|
Storage
|
| 8068a |
1
|
Staining solution A |
1 ml
|
4°C
|
| 8068b |
1
|
Staining solution B |
1ml
|
4°C
|
| 8068c |
1
|
Staining solution C |
0.2 ml
|
4°C
|
| 8068d |
1
|
Staining solution D |
20 ml
|
4°C
|
| 8068e |
1
|
X-Gal solution |
5 ml
|
-20°C
|
| 8068f |
1
|
100X Fixing solution |
1 ml
|
4°C
|
Quality Control
Human umbilical vein endothelial cells (HUVECs) are transfected
with Promega rhoSV-bata-galactosidase control vector. Beta-Galactosidase
Colorimetric Assay is applied to assay the gene expression 24 hours
post transfection, as shown in Figure 1.
Procedure
A. Preparation of reagents
1. Preparation of working fixing solution: Prepare working fixing
solution by diluting 100× Fixing Solution stock 1:100 in PBS.
2. Preparation of working staining solution: Prepare fresh working
staining solution based on the number of samples to be assessed.
For each sample in 35 mm plate, prepare the following mixture:
100 µl of X-gal Solution
20 µl of Staining Solution A
20 µl of Staining Solution B
4 µl of Staining Solution C
400 µl of Staining Solution D
1456 µl of DI H2O
Total of 2000 µl of working staining solution
B. Staining protocol
1. Remove culture medium from cells and rinse cells twice with PBS.
2. Fix cells by incubating with 2 ml of working fixing solution
for 3-5 minutes at room temperature.
3. Aspirate fixing solution and rinse the fixed cells three times
with PBS.
4. Add 2 ml of working staining solution to the cells and incubate
overnight at 37°C, the blue color should develop in beta-galactosidase
expressing cells.
5. Remove working staining solution and rinse cells twice with PBS,
store cells in PBS at 4°C until examination under light microscope.
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