X Gal/Beta Galactosidase Assay
/***********************************************************************************************************************/
X-Gal/Beta-galactosidase assays
Solutions
Z Buffer
0.75 g KCl
16.1 g Na2HPO4 7H2O
0.246 g MgSO4 7H2O
5.5 g NaH2PO4 H2O
Bring the final volume to 1 liter using ddH2O, adjust pH to 7 and store at 4deg C.
Alternatively, for 500mls, using these reagents:
0.375 g KCl
4.27 g Na2HPO4 H2O
0.061 g MgSO4 H2O
2.75 g NaH2PO4 H2O
Bring the final volume to 500 mL using ddH2O, adjust pH to 7 and store at 4deg C.
Z Buffer with 2-Mercaptoethanol:
Add 270 μl of 2-Mercaptoethanol per 100 ml of Z Buffer, just prior to use.
X-gal, 100X stock solution:
100mg/ml in Dimethyl formamide
Add X-gal stock solution to Z Buffer with 2ME, to a final concentration of 1mg/ml, prior to use.
Procedure
1. For colony lifts from solid agar, nitrocellulose filters were used. After cutting an appropriately sized filter to fit the surface of a petri plate, nitrocellulose was laid on the agar surface and then removed using tweezers.
2. The filters were then placed onto an aluminum sheet that was floated on liquid nitrogen, for approx. 20 seconds. The filters were then immersed in liquid nitrogen for 2 seconds, thawed at room temperature.
3. Filter lifts were then placed cell side up onto pads of Whatman #1 filter paper, in empty petri-plates, that had been soaked with Z-buffer solution containing 2-mercaptoethanol and X-gal.
4. The filter lifts were then incubated at 30 C for 30min to overnight, until the development of a blue precipitate was clearly visible.
5. A similar approach was used in the semi-quantitative assays. Alternatively, yeast cells from liquid culture were immobilized onto nitrocellulose filter sheets using a Bio-Rad “Bio-Dot” apparatus connected to a vacuum manifold. The immobilized cells were lysed using liquid nitrogen and incubated with x-gal containing buffer as outlined above.
6. For the analysis of beta-galactosidase expression in cells grown in 96 well micro-titer dishes, cotton applicators were used to remove cells from each well and then immersed directly into liquid nitrogen. After thawing at room temperature, they were then incubated for ~30-overnight in wells containing 150 μL of X-gal buffer.
References:
Cell, Vol. 74, 205-214, July 16, 1993
Mammalian Ras interacts directly with the serine/threonine kinase raf.
Anne B. Vojtek, Stanley M. Hollenberg and Jonathan A. Cooper.
A very good overview of various beta-galactosidase activity assays performed in yeast:
http://www.foxchase.org/research/labs/golemis/betagal/beta_gal_yeast.htm
|
"
