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B-galactosidase assay

Reagents:

1. 100X Mg Buffer- 10 ul 1M MgCl₂
   35 ul BME (make up fresh)
   55 ul H20
   
   
2. 0.8 M Phosphate Buffer, pH 7.5- NaH₂PO₄-H₂0 (M.W. 120)- 4.42 g
   Na₂HPO₄ (M.W. 141.96)- 23.86 g (store at 25°C)
   Make volume up to 250 ml with ddH20
   
   
3. 1X ONPG (o-NITROPHENYL b-D-GALACTOPYRANOSIDE Sigma Cat. N 1127)-
   4 mg/ml. Resuspend in 0.1M Phosphate Buffer (store at -20°C)
   
   
4. 1 M Na₂CO₃ STOP Solution (store at 25°C)
   
   
5. b-galactosidase- (Grade VIII: purified from E. Coli Sigma Cat. G 5635)

Protocol:

1. To each sample add the following: 30 ul cell extract (adjusted to constant protein amt)
   66 ul 1X ONPG
   3 ul Mg Buffer
   201 ul 0.1 M Phosphate Buffer (diluted 1:8 from stock)
   
   
2. Prepare positive control: 1 ul b-galactosidase enzyme
   66 ul ONPG
   3 ul Mg Buffer
   229 ul 0.1 M Phosphate Buffer
   
   
3. Prepare blank/neg. control: 66 ul ONPG
   3 ul Mg Buffer
   230 ul 0.1 M Phosphate Buffer
   
   
4. Incubate at 37°C until a faint yellow color is visible in the tubes (this can be 15 min. to overnight depending on the efficiency of transfection)
   
   
5. Read at E410 on the spectrophotometer (the assay is linear from .010-1.00)

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