b-GALACTOSIDASE STAINING

(For dishes, well 35 mm)

 

1- Rinse cells 2x with 1X PBS

2- Add 1 ml of 0.5% glutaraldehyde in PBS

3- Let at room temperature for 15 min.

4- Rinse 3x with PBS (3-5 min. each time)

5- Add 1 ml of Stain Solution

6- Stain 1 hour to O/N at 37°C incubator

7- Count cells under microscope for X-gal staining

 

0.5% glutaraldehyde in PBS

= 1/50e of 25% glutaraldehyde

= 200 ml of 25% glutaraldehyde in 10 ml PBS

Stain Solution = 5 mM K Ferricyanide, 5 mM K Ferrocyanide, 1 mM MgSO4, 1 g/ml X-gal

K Ferricyanide 12.5 ml

K Ferrocyanide 12.5 ml

1 M MgSO4 1 ml

50 mg/ml X-gal 20 ml

Qs 1X PBS to 1 ml

DOSAGE b-GALACTOSIDASE

(Protocole M. Darmon given by Alexi)

 

1- Break cells:

- Rinse cells 1x or 2x with 1X PBS

- Add at the middle of the dish 150 ml of 250 mM sucrose, 10 mM Tris

- Scrape cells

- Transfer in eppendorf tube

- Freeze in Liquid Nitrogen

Thaw at 37°C in a water-bath

Vortex 1 to 2 min. to break cells

- Repeat this step

- Centrifuge at 12,000 rpm for 1 min.

2- Transfer supernatant in closed hemolysis tube

Add to 1.2 ml of solution:

Z Buffer 1 ml

ONPG (4mg/ml in Z Buffer) 0.2 ml

b-mercapto EtOH 3.3 ml

3- Incubate at 37°C in a water-bath until apparation of yellow color

4- Stop reaction with 0.5 ml 1 M Sodium Carbonate

Note time

Read at 420 nm (Blank = solution Z Buffer/ONPG/b-mercapto EtOH)

5- Express the results in Units/dish and/or Units/mg/proteins

 

 

Buffers

Z Buffer = 50 mM Na2HPO4, 40 mM NaH2PO4, 10 mM KCl, 1 mM MgCl2

Na2HPO4 2.13 g

NaH2PO4 1.66 g

1 M KCl 3 ml

1 M MgCl2 0.3 ml

Qs H2O to 300 ml

 

ONPG solution

4 mg/ml in Z Buffer (0.8 mg/tube of dosage)

---> Solubilize at 37°C for 30 min.