LacZ staining of cells

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The bacterial enzyme β-galactosidase (gene LacZ) is frequently used as reporter gene. It can be easily located with a LacZ stain using the artificial substrate X-gal, which turns blue when it is cleaved by β-galactosidase.

This protocol describes the staining of cultured cells containing active LacZ genes.

material

  • X-gal stock is 200 mg/ml in dimethylformamide (DMF)
  • K ferri-cyanide ( 50mM ) =Fe3- is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131)
  • K ferro-cyanide ( 50mM ) =Fe4- is 0,42 g / 20 ml PBS ( Sigma Cat.# P-9387)

staining solution

  • make fresh
  • heat mix to 50°C before adding X-Gal, in order to avoid precipitation
10 ml 15 ml 30 ml 50 ml
X-gal ( 200 mg/ml) 50 µl 75 µl 150 µl 250 µl
MgCl2 (1M) 20 µl 30 µl 60 µl 100 µl
K ferri-cyanide ( 50mM ) 1 ml 1,5 ml 3 ml 5 ml
K ferro-cyanide ( 50mM ) 1 ml 1,5 ml 3 ml 5 ml
PBS ( up to...) 7,9 ml 8,5 ml 23,5 ml 39,6 ml

fixation solution

  • make fresh
10 ml 15 ml 30 ml 50 ml
formaldehyde/methanal ( 37 %) 541 µl 811,5 µl 1,62 ml 2,7 ml
glutaraldehyde ( 25 %) 40 µl 60 µl 120 µl 200 µl
PBS (up to...) 9,42 ml 14,13 ml 28,26 ml 47,1 ml

common mistakes / tips

  • time of fixation is crucial; overfixing cells will reduce LacZ activity
  • some cell types are easy to detach by squirting liquid onto the dish; be careful;
  • X-Gal will precipitate if solution cools too much

steps

  1. carefully wash cells once with PBS (avoid detaching cells by strong pipetting)
(volume is around 5 ml for 10 cm-dish
                  3 ml for 6 cm-dish			
                 500µl for a 24 well)		
  1. add fixative solution
  2. incubate 2 min (Time is important !)
  3. carefully wash 3 times with PBS (1st wash must be quick to avoid overfixation in the last samples to be washed)
  4. add staining solution
  5. incubate over night at 37°C

sample stains

see also