LacZ staining of cells

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{{back to protocols}}
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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]].
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This protocol describes the staining of cultured cells containing active LacZ genes.
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== material ==
== material ==
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* X-Gal stock is 200 mg/ml in dimethylformamide (DMF)
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* X-gal stock is 200 mg/ml in dimethylformamide (DMF)
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* K ferri-cyanide ( 50mM )=K3 is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131)
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* K ferri-cyanide ( 50mM ) =Fe<sup>3-</sup> is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131)
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* K ferro-cyanide ( 50mM )=K4 is 0,42 g / 20 ml PBS ( Sigma Cat.# P-9387)
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* K ferro-cyanide ( 50mM ) =Fe<sup>4-</sup> is 0,42 g / 20 ml PBS ( Sigma Cat.# P-9387)
=== staining solution ===
=== staining solution ===
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| MgCl<sub>2</sub> (1M)||20 µl||30 µl||60 µl||100 µl
| MgCl<sub>2</sub> (1M)||20 µl||30 µl||60 µl||100 µl
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|-
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| K ferri-cyanide ( 50mM )=K3||1 ml||1,5 ml||3 ml||5 ml
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| K ferri-cyanide ( 50mM )||1 ml||1,5 ml||3 ml||5 ml
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|-
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| K ferro-cyanide ( 50mM )=K4||1 ml||1,5 ml||3 ml||5 ml
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| K ferro-cyanide ( 50mM )||1 ml||1,5 ml||3 ml||5 ml
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|-
| PBS ( up to...)||7,9 ml||8,5 ml||23,5 ml||39,6 ml
| PBS ( up to...)||7,9 ml||8,5 ml||23,5 ml||39,6 ml
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* [http://axon.med.harvard.edu/~cepko/protocol/xgalplap-stain.htm LacZ reaction, whole mount, tissue section staining protocol]
* [http://axon.med.harvard.edu/~cepko/protocol/xgalplap-stain.htm LacZ reaction, whole mount, tissue section staining protocol]
* [http://en.wikipedia.org/wiki/Lacz LacZ background from wikipedia]
* [http://en.wikipedia.org/wiki/Lacz LacZ background from wikipedia]
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* [[Beta-Galactosidase Assay (A better Miller)]]
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* [[Beta-Galactosidase Assay (A better Miller)]], [[Beta-galactosidase assay]]
* [[BE.109:Protein engineering/Assessing beta-galactosidase]]
* [[BE.109:Protein engineering/Assessing beta-galactosidase]]
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* [[X-Gal]]
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[[Category:Protocol]]
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[[Category:Protein]]

Current revision

back to protocols

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.

Contents

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 ml15 ml30 ml50 ml
X-gal ( 200 mg/ml)50 µl75 µl150 µl250 µl
MgCl2 (1M)20 µl30 µl60 µl100 µl
K ferri-cyanide ( 50mM )1 ml1,5 ml3 ml5 ml
K ferro-cyanide ( 50mM )1 ml1,5 ml3 ml5 ml
PBS ( up to...)7,9 ml8,5 ml23,5 ml39,6 ml

fixation solution

  • make fresh
10 ml15 ml30 ml50 ml
formaldehyde/methanal ( 37 %)541 µl811,5 µl1,62 ml2,7 ml
glutaraldehyde ( 25 %)40 µl60 µl120 µl200 µl
PBS (up to...)9,42 ml14,13 ml28,26 ml47,1 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

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