Notes

1 AEBSF, a water-soluble serine protease inhibitor, may be used instead of PMSF

2 Alternative lysis buffers and lysis protocols may be used, particularly if assays for other cotransfected reporter genes require specific lysis buffers. The performance of alternative buffers should be compared with the Galacto-Light lysis solution to ensure optimum results

3 Reducing agents such as p-mercaptoethanol or DTT will decrease the half-life of light emission of Galacton and particularly Galacton-Plus If the extended halflife of light emission from Galacton-Plus is crucial to the assay, reducing agents should be omitted from the lysis solution. If they cannot be omitted, they should be minimized and care should be taken to ensure that the same concentration of reducing agent is present in each assay tube or well This will ensure that the kinetics of light emission from each assay do not vary If lysis buffer containing DTT has been used, the addition of hydrogen peroxide to the light emission accelerator to a final concentration of 10 mM (add 1 pL of 30% H202/1 mL of light emission accelerator) will prevent rapid decay of signal half-life

4 The use of glass tubes for preparation of yeast extracts results in more efficient cell breakage than with microcentrifuge tubes Glass beads are used directly from the bottle, without washing or reconstitution To measure, mark desired volume on pipet tip, and scoop beads to level of mark

5 Yeast cell extracts can be stored at -70°C; however, it is optimal to store cell pellets at—70°C instead and prepare extracts just before use

6 Most cells and tissues have some level of endogenous P-galactosidase activity Significant reductions of endogenous enzyme activity can be achieved by selective heat inactivation (18) Endogenous enzyme activity is reduced at the pH of the reaction buffer, whereas the transfected bacterial enzyme activity is only slightly affected (5). It is important to determine the level of endogenous enzyme activity in extract from nontransfected cells to establish assay background.

7 The amount of cell extract required may vary depending on the level of expression and the instrumentation used Use 5 pL of extract for positive controls and 10—20 pL of extract with cells containing a potentially low level of enzyme, however, it is essential to maintain a constant volume of extract and/or lysis solution in each reaction, since the DTT in the lysis solution can affect the chemilumines-cent reaction It is important to vary the amount of extract to keep the signal within the linear range of the assay

8 Reaction component volumes can be scaled down to accommodate a microplate assay format or a luminometer with a smaller volume injector, although sensitivity may be affected slightly. It is recommended to keep the volume of cell extract 5-20 pL.

9 The incubation period with reaction buffer may be as short as 15 min, but the dynamic range of the assay may decrease For yeast extracts, it is recommended that this incubation be only 15 min After a 20-min incubation, the reaction may lose linearity, most likely, owing to proteolysis (D Nathan and S Lindquist, personal communication) For tissue extracts, a 10 min incubation has been used (11)

10 Light intensities are time dependent Reaction buffer should be added to samples within the same time frame as they are measured in the luminometer For example, if it takes 10 s to complete a measurement, then reaction buffer should be added to tubes every 10 s

11 If manual injection is used, then the light emission accelerator should be added in the same consistent time frame as the addition of reaction buffer. This is critical when using Galacton. Galacton substrate has a half-life of light emission of approximately 4 5 min after the addition of light emission accelerator Galacton-Plus substrate emits light with a significantly longer half-life {iV2 =180 min) after the addition of light emission accelerator

12. A liquid scintillation counter may be used as an alternative to a luminometer (19,20), however, the resulting assay sensitivity may be lower When light emission reaches a maximum, the changes in signal intensity per unit time are at a minimum (steady-state or approaching steady-state) Therefore, the signal should be measured during this period Some scintillation counters permit a measurement of chemiluminescence directly by turning off the coincidence circuit. If chemiluminescence is measured without turning off this circuit, a linear relationship between the light level and scintillation counts can be established by taking the square root of the counts per minute minus instrument background (19).

Actual = (measured-instrument background)172

13. Alternate lysis buffers may be used, however, we recommend that their performance is compared with the GUS lysis buffers to ensure optimum results. A lysis buffer compatible with the luciferase assay, containing QAM potassium phos phate, 1 mM DTT, and 1 mg/mL BSA has equivalent performance to the GUS lysis buffer

14 Bacterial contamination of plant material will cause high background Best results will be obtained with sterile preparations Chlorophyll in concentrated samples may interfere with signal intensity Therefore, if high levels of chlorophyll are present, several dilutions of extract should be assayed

15 The incubation with GUS reaction buffer may be as short as 15 min (especially if high levels of expression are expected), but the dynamic range of the assay may decrease. Light intensities are time dependent Reaction buffer should be added to samples within the same time frame as they are measured in the lummometer For example, if it takes 10 s to complete a measurement, then reaction buffer should be added to tubes every 10 s.

16 If manual injection is used, then the light emission accelerator should be added in the same consistent time frame as the addition of reaction buffer.

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