β-Galactosidase Staining Kit

Catalog No. ABIN3198655

Product Details

Application: Biochemical Assay (BCA)

Sample Type: Cell Samples

Characteristics: The β-galactosidase staining kit provides an easy-to-use and efficient method to determine the transfection efficiency and expression of LacZ gene. β-galactosidase catalyzes the hydrolysis of X-gal, which produces a blue color in cells expressing the transfected gene. Each kit provides sufficient quantities to perform up to 75 assays in 35 mm dishes.

Components:

1. 100X Fixing Solution : One tube - 1.5 mL of 25% Glutaraldehyde
2. Staining Solution A : One tube - 1.5 mL of 500 mM Potassium Ferrocyanide
3. Staining Solution B : One tube - 1.5 mL of 500 mM Potassium Ferricyanide
4. Staining Solution C : One tube - 1.5 mL of 200 mM MgCl2
5. X-gal Solution : Three tubes - 1.5 mL of 40mg/mL X-gal in DMF for each tube

Material not included:

1. PBS
2. 37 °C Incubator
3. Light microscope
4. Cells or tissue samples expressing LacZ

Application Details

Application Notes: Optimal working dilution should be determined by the investigator.

Comment:

• Measures the transfection efficiency of the LacZ gene
• Simple staining protocol
• Sufficient reagents for 75 assays in 35 mm culture dishes

Reagent Preparation:

1X Fixing Solution: Prepare a 1X Fixing Solution by diluting the provided 100X stock 1:100 in 1X PBS. Store the diluted solution at room temperature for up to six months. 2 Cell Staining Working Solution: Prepare FRESH cell staining working solution based on the number of samples. The chart below is suggested for samples in 35 mm plate, and may be modified accordingly to suit other culture plate sizes. 10 plate (35 Reagents 1 plate (35 mm) 5 plate (35 mm) mm) Staining Solution A 20 μL 100 μL 200 μL Staining Solution B 20 μL 100 μL 200 μL Staining Solution C 20 μL 100 μL 200 μL X-Gal Solution 50 μL 250 μL 500 μL 1X PBS 1.89 mL 9.45 mL 18.9 mL Total 2 mL 10 mL 20 mL Staining Protocol for a 35 mm Plate 1. Aspirate the medium from the LacZ gene transfected or infected cells. 2. Wash the cells twice with 3 mL of 1X PBS and aspirate the final wash. 3. Add 2 mL of 1X Fixing Solution. Incubate at room temperature for 5 minutes. 4. Remove the fixing solution and wash the fixed cells three times with 3 mL of 1X PBS. 5. Aspirate the final wash, and completely cover cells by adding 2 mL of freshly prepared Cell Staining Working Solution. 6. Incubate the cells at 37 °C protected from light for 1 hr to overnight. 7. Remove the Cell Staining Working Solution, then wash the stained cells twice with 3 mL of 1X PBS and store cells in 1X PBS. For long-term storage, overlay the cells with 1X PBS containing 20 % Glycerol. Store at 4 °C. Note: Excess amount of salt crystals can be removed by briefly incubating the stained sample with DMSO. 8. Count the blue stained cells using light microscope. To determine transfection or infection efficiency, calculate the ratio of blue stained cells to total cells.

Restrictions: For Research Use only

Handling

Storage: 4 °C/-20 °C

Storage Comment: Store X-gal solution protected from light at -20°C, and other kit components at 4°C.

References

Rani, Bhardwaj, Srivastava, Sharma, Parsad, Kumar: "Senescence in the lesional fibroblasts of non-segmental vitiligo patients." in: Archives of dermatological research, Vol. 309, Issue 2, pp. 123-132, (2017) (PubMed).

Xu, Zheng, Bian, Xie, Liu, Zhen, Crane, Zhou, Cao: "Aberrant Activation of TGF-? in Subchondral Bone at the Onset of Rheumatoid Arthritis Joint Destruction." in: Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research, Vol. 30, Issue 11, pp. 2033-43, (2015) (PubMed).

Ge, Pettan-Brewer, Morton, Carter, Fatemi, Rabinovitch, Ladiges: "Mitochondrial catalase suppresses naturally occurring lung cancer in old mice." in: Pathobiology of aging & age related diseases, Vol. 5, pp. 28776, (2015) (PubMed).

Black, Trackman et al.: "Transforming growth factor-beta1 (TGFbeta1) stimulates connective tissue growth factor (CCN2/CTGF) expression in human gingival fibroblasts through a RhoA-independent, Rac1/Cdc42-dependent mechanism: ..." in: The Journal of biological chemistry, Vol. 283, Issue 16, pp. 10835-47, (2008) (PubMed).

Target Details

Background: The delivery of reporter genes into cells has become the primary means by which researchers study gene function and gene expression regulation. LacZ is a commonly used reporter gene in transfection experiments because the gene product, β-galactosidase, is very stable and resistant to proteolytic degradation and easily assayed.