Calcium-Binding Protein Protocols Calcium-Binding Protein Protocols

Gene Expression in Transfected Cells 359
11. Using a sterile Pasteur pipet, gently transfer all of the 0.5-mL transfected cells,
including a white aggregate of cells that forms as a result of the electroporation,
to the cell-culture flask containing 10 mL prewarmed medium from step 1. Be
sure to transfer all of the cells by rinsing the cuvet with medium.
12. Incubate the transfected cells in the 37°C carbon dioxide incubator for 8–72 h
(see Note 7). During the course of a long incubation, fast-growing cells may have
to be diluted to prevent the culture from becoming too dense.
3.2. Lysis of the Cells
1. Harvest the 10.5 mL of transfected cells from Subheading 3.1., step 12 in 15-mL
tubes by centrifugation at 250g for 10 min at room temperature.
2. Remove the supernatant. Resuspend the cells in 1 mL PBS and transfer them to
1.5-mL tubes. Centrifuge at 250g for 10 min at room temperature.
3. Remove the supernatant and resuspend the cell pellet in 100 µL of lysis buffer by
gentle pipetting. Incubate for 5 min at room temperature. At this stage, the cell
lysates can be stored at –70°C, or alternatively proceed directly to the assays.
3.3. β-Galactosidase and Luciferase Assays
Centrifuge the lysed cells from Subheading 3.2., step 3 at 18,000g for 1 min
at room temperature to pellet the cell debris. Place the tubes on ice and keep
them on ice for the rest of the analysis.
In order to compare different transfections with each other, the enzyme
activity present in the same volume of lysate from each transfection has to
be compared. However, the β-galactosidase can be measured from one volume
and the luciferase from a different volume.
3.3.1. β-Galactosidase Assay
1. β-galactosidase catalyses the hydrolysis of ONPG to o-nitrophenol, which is yellow.
Aliquot 250 µL ONPG solution (0.8 mg/mL in β-galactosidase assay buffer)
to 1.5-mL tubes, preparing one tube more than the number of transfections.
2. Add 20 µL of the supernatant of each centrifuged lysate to a tube with ONPG
solution. Mix well.
3. Incubate at room temperature until the samples are light yellow. Depending on
the cell line, this will take anywhere from 5 min to overnight. If no yellow color
appears overnight, your transfection has been unsuccessful. If this is the case,
repeat Subheading 3.1. with different conditions (see Notes 3–6).
4. To directly compare the amount of β-galactosidase activity in each sample, stop
the reactions of all samples after the same incubation time. When yellow color is
reached in all samples, stop the reactions by adding 250 µL of 1 M Na 2CO 3. Also
add 250 µL of 1 M Na 2CO 3 to the extra “blank” tube prepared in step 1. Mix well.
5. Measure the optical density at 420 nm (OD 420) of the reactions vs the “blank.”
The β-galactosidase-catalyzed reaction is linear and can be accurately measured
between an OD 420 of 0.2 and 0.8. If the OD 420 of your samples lie outside of this