Protocol for b-Galactosidase

1. INTRODUCTION

The study of the lac operon has played an important role in understanding the control of gene expression in bacteria. In prokaryotes, gene expression is controlled primarily at the level of transcription. For eukaryotes, the promoter activity can be analyzed by using fusion genes containing the promoter of interest attached to the bacterial b -galactosidase gene. The level of b -galactosidase expression indicates the level of transcription under different regulatory conditions.

4-methylumbelliferone provides a sensitive, quantitative assay for b -galactosidase. Cleavage of 4-methylumbelliferyl-b -D-galactoside by b -galactosidase yields the fluorescent molecule 4-methylumbelliferone (7-hydroxy-4-methylcoumarin, 4MU). 4-methylumbelliferone is fluorescent above pH 8. When excited by 365nm light, 4MU emits light at 460nm. Used with the Turner BioSystems TD-700 Laboratory Fluorometer, the assay is sensitive enough to detect picogram levels of b -galactosidase.

2. MATERIALS REQUIRED

• TD-700 Laboratory Fluorometer with standard PMT and 10mm x 10mm square cuvette adaptor (P/N 7000-009).
• Near UV lamp (P/N 10-049).
• Long Wavelength UV Filter Kit (P/N 10-302R) which includes 300-400 nm excitation filter (P/N 10-069R) and emission filter (P/N 10-110R-C).
• 10mm x 10mm square methacrylate disposable cuvettes (P/N 7000-959).
• Microcentrifuge
• 4-methylumbelliferone, sodium salt, MW=198.20
• Distilled water
• Sodium carbonate, anhydrous, MW=105.99
• 133 mM Glycine
• 25 mM Tris-HCl, pH 7.5
• 125 mM NaCl
• 2 mM MgCl₂
• 12 mM 2-mercaptoethanol
• 0.3 mM 4-methylumbelliferyl-b -D-galactoside (0.1 mg/ml)
• 25% (w/v) trichloroacetic acid

3. 4MU SOLUTION PREPARATION

3.1 4MU stock solution A (1mM):

19.8mg 4-methylumbelliferone
Add distilled water to 100ml
Store at 4^°C, away from light.

3.2 4MU stock solution B (1µM):

10 µl 4MU stock solution A
Dilute with 10 ml distilled water.
Store at 4^°C, away from light.

3.3 Carbonate stop buffer (0.20M):

21.2g Sodium carbonate, anhydrous, MW = 105.99
Add distilled water to 1000ml.

3.4 Glycine-Carbonate stop buffer

133mM glycine
83mM Na₂CO₃
pH to 10.7

3.5 4-Methylumbelliferone standard, 50nM:

100.0 µl 1 uM 4MU solution
1.9 ml Glycine-Carbonate stop buffer
Prepare just before use.

3.6 Reaction Cocktail:

25 mM Tris-HCl, pH to 7.5
125 mM NaCl
2 mM MgCl₂
12 mM 2-mercaptoethanol
0.3 mM 4-methylumbelliferyl-b -D-galactoside (0.1 mg/ml) substrate. Prepare reaction cocktail minus substrate at room temperature. Disperse substrate into a small volume of ethanol (0.5% of the total volume of cocktail). Add ethanol- substrate solution to acqueous cocktail. Then rapidly vortex until dissolved.

3.7 TCA solution:

25% (w/v) trichloracetic acid

4. CALIBRATION PROTOCOL

NOTE: Accurate pipetting and thorough mixing are critical for reproducible results. However, take extreme care when mixing samples; do not introduce air bubbles. Air bubbles can cause scattering of light leading to inaccurate results. If air bubbles form, hold the upper portion of the cuvette in one hand and gently tap the bottom sides of the cuvette with your other hand to release bubbles.

4.1 Install filter 10-069R excitation and emission filter 10-110R-C. For further assistance refer to Section III (Optical Filter Installation and Removal) on page 9 of the TD-700 Operating Manual.

4.2 Insert the Near U.V. Mercury Vapor lamp (P/N 10-049). Then turn on the instrument and allow a 10 minute warm-up period. For further assistance refer to Section IV (Lamp Installation and Removal) on page 11 of the TD-700 Operating Manual.

4.3 Select units of measurement. Press <MENU>, <2>, and select desired units.

4.4 Prepare 2ml volumes of 50, 100, 150, and 200 nM 4MU standards in Glycine-Carbonate stop buffer.

4.5 Calibrate the instrument with standard prepared from step 4.2. Mix well. Insert cuvette containing standard into the instrument, close the lid, and press <CAL>. Enter in the actual concentration of the standard or a convenient value that will display a multiple of the actual DNA concentration, then <ENT>. If a value other than the actual concentration is used, be sure to note the multiplication factor used. Remove cuvette.

NOTE: Generating a standard curve verifies the linearity of the assay within a particular concentration range. It is recommended that you perform this at least once when working with a new instrument or performing the assay for the first time. Also, you may want to generate a standard curve every few weeks as a quality check on the standard, a reliability check on the instrument, and a consistency check on technique.

4.6 Blank the instrument with Glycine-Carbonate stop buffer. Place 1.9 ml assay solution into a cuvette, insert into the instrument, close the lid, and press <BLANK>. Press <1> to accept blank value. Remove cuvette.

5. ASSAY

5.1 Add 40 µl of sample to a centrifuge tube. Use water for a blank.

5.2 Add 160 µl of the reaction cocktail.

5.3 Incubate at 37⁰C for 30 minutes.

5.4 Stop the reaction by adding 50 µl of 25% TCA. Cool on ice.

5.5 Clarify the solution by centrifugation in a microcentrifuge for 1 to 2 minutes.

5.6 Add 100 µl of supernatant to 1.9 ml of Glycine-Carbonate reagent.

5.7 Read the fluorescence by inserting the cuvette into the fluorometer, closing the lid, and pressing <GO>. Determine the concentration from the standard curve in section 4.

6. NOTES

6.1 A 40 µl sample should contain 10^-6 to 10^-5 units (nM 4MU min^-1) of b -galactosidase activity. If other dilutions of the sample into the reaction cocktail are used, adjust the samples accordingly.

6.2 The Glycine-Carbonate reagent is sufficient to titrate 1/3 its own volume in 5% TCA to the proper pH for reading (200 µl reaction stopped with 50 µl of 25% TCA).

6.3 The protocol is designed for assay of E.coli b -galactosidase activity, which is active at neutral pH. The vertebrate form of b -galactosidase is a lysosomal enzyme, which has optimal acitivity at pH 4.5. The lysosomal activity can be assayed in acetate buffer instead of Tris buffer. The assay should be performed at both pH values when lysosomal contamination of reactions is suspected.

6.4 Depending on the calibration setting used, maximum readings are obtained at 40 to 200nM 4MU final concentration in the Glycine-Carbonate buffer.

6.5 Limitations on the sensitivity of the assay are determined by the background fluorescence of the substrate. It is therefore important to use freshly prepared substrate solutions in assays where high sensitivity is desired.

6.6 Frozen reaction cocktails may be used, but backgrounds gradually increase with repeated freeze/thaw cycles. In addition, substrate sometimes precipitates from frozen cocktails. Resolubilization is slow unless the cocktail is heated to 37^°C and repeatedly vortexed.

7. ABOUT Turner BioSystems, INC.

Orders for Turner BioSystems products may be placed by:

Phone: (408) 749-0994 or Fax: (408) 749-0998
E-mail: sales@turnerbiosystems.com
Internet: www.turnerbiosystems.com

Mailing Address (HQ): Turner BioSystems, Inc.
845 W. Maude Avenue
Sunnyvale, CA 94085