Beta-Galactosidase Colorimetric Assay

Catalog # 8068
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Description

50 Tests in 35mm plate

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 ScienCellT 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
# of Vials
Reagents
Quantity
Storage
8068a 1 Staining solution A 1 ml 40C
8068b 1 Staining solution B 1 ml 40C
8068c 1 Staining solution C 0.2 ml 40C
8068d 1 Staining solution D 20 ml 40C
8068e 1 X-Gal solution 5 ml -200C
8068f 1 100X Fixing solution 1 ml 40C

Quality Control

Human umbilical vessel endothelial cells (HUVECs) are transfected with Promega® ?SV-bata-galactosidase control vector. ß-Galactosidase Colorimetric Assay is applied to assay the gene expression 24 hours post transfection, as shown in Figure 1.

Procedures

  1. 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
      +  1856 µl of Staining Solution D
      -------------------------------------------
        2000 µl of working staining solution
  2. 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 ß-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.

* Crystal deposition, which comes from unreacted X-gal, may be observed after incubation of cells with working staining solution. It can be minimized by pre-filtering the working staining solution with a 0.2 µm filter