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 ) =Fe^3- is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131)
• K ferro-cyanide ( 50mM ) =Fe^4- 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

• 

  weak LacZ stain from modified mouse embryonic stem cells, 200x

• 

  strong LacZ stain from modified mouse embryonic stem cells, 100x

see also

• LacZ reaction, whole mount, tissue section staining protocol
• LacZ background from wikipedia
• Beta-Galactosidase Assay (A better Miller), Beta-galactosidase assay
• BE.109:Protein engineering/Assessing beta-galactosidase
• X-Gal