LacZ staining of cells: Difference between revisions
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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 == | == material == | ||
* X- | * X-gal stock is 200 mg/ml in dimethylformamide (DMF) | ||
* K ferri-cyanide ( 50mM )= | * K ferri-cyanide ( 50mM ) =Fe<sup>3-</sup> is 0,33 g / 20 ml PBS ( Sigma Cat.# P-8131) | ||
* K ferro-cyanide ( 50mM )= | * K ferro-cyanide ( 50mM ) =Fe<sup>4-</sup> is 0,42 g / 20 ml PBS ( Sigma Cat.# P-9387) | ||
=== staining solution === | === staining solution === | ||
Line 18: | Line 24: | ||
| 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 | ||
|- | |- | ||
| K ferri-cyanide ( 50mM ) | | K ferri-cyanide ( 50mM )||1 ml||1,5 ml||3 ml||5 ml | ||
|- | |- | ||
| K ferro-cyanide ( 50mM ) | | 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 | | PBS ( up to...)||7,9 ml||8,5 ml||23,5 ml||39,6 ml | ||
Line 58: | Line 64: | ||
# add staining solution | # add staining solution | ||
# incubate over night at 37°C | # incubate over night at 37°C | ||
== sample stains == | |||
<gallery> | |||
Image:XGal_weak_ESC_200x.jpg|weak LacZ stain from modified mouse embryonic stem cells, 200x | |||
Image:XGal_strong_ESC_100x.jpg|strong LacZ stain from modified mouse embryonic stem cells, 100x | |||
</gallery> | |||
== see also == | == see also == | ||
* [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] | ||
* [[Beta-Galactosidase Assay (A better Miller)]] | * [[Beta-Galactosidase Assay (A better Miller)]], [[Beta-galactosidase assay]] | ||
* [[BE.109:Protein engineering/Assessing beta-galactosidase]] | * [[BE.109:Protein engineering/Assessing beta-galactosidase]] | ||
* [[X-Gal]] | |||
[[Category:Protocol]] | |||
[[Category:Protein]] |
Latest revision as of 08:39, 10 June 2008
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.
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
- 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)
- add fixative solution
- incubate 2 min (Time is important !)
- carefully wash 3 times with PBS (1st wash must be quick to avoid overfixation in the last samples to be washed)
- add staining solution
- 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