b-Galactosidase Staining Kit
Catalog No. 35001(version B)

Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, California 92008, USAToll free:877 222 9543
Telephone:
760 431 1263
Fax:
760 431 1351
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone: 0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone:		+32 (0)2 653 0001
Fax:		
+32 (0)2 653 0050
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone:+81 3 5225 3638
Fax:+81 3 5261 8733
Copyright 2009 Active Motif, Inc.

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Information in this manual is subject to change without notice and does not constitute a commitment on the part of Active Motif, Inc. It is supplied on an “as is” basis without any warranty of any
kind, either explicit or implied. Information may be changed or updated in this manual at any time.
This documentation may not be copied, transferred, reproduced, disclosed, or duplicated, inwhole or in part, without the prior written consent of Active Motif, Inc. This documentation isproprietary information and protected by the copyright laws of the United States and international treaties.
The manufacturer of this documentation is Active Motif, Inc.
© 2009 Active Motif, Inc., 1914 Palomar Oaks Way, Suite 150; Carlsbad, CA 92008. All rights reserved.
All trademarks, trade names, service marks or logos referenced herein belong to their respectivecompanies.
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TABLE OF CONTENTS

PageOverview  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Kit Components and Storage
Additional Materials Required . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
Protocolsb-Galactosidase Staining Kit  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
Appendix
Section A. Troubleshooting Guide . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3
Section B. Calculation Of Efficiency . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .3
Technical Services  . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4

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Overview
The b-galactosidase Staining Kit provides an easy-to-use and efficient method to determine thepercentage of cells expressing lacZ following transient or stable transfection. The gene product oflacZ, b-galactosidase, catalyzes the hydrolysis of X-gal, which produces a blue color that iseasily visualized.
This kit can also be used to determine the efficiency of direct delivery of b-galactosidase protein
by Chariot™ Protein Delivery Reagent (Catalog Nos. 30025 & 30100).

productformat

catalog no.

b-Galactosidase Staining Kit

75 rxns

35001

Kit Components and Storageb-Galactosidase Staining Kits are for research use only. Not for use in diagnostic procedures.
All components are guaranteed stable for 6 months from date of receipt when stored properly.
Reagents

Quantity

Composition

Storage

Staining Solution 1

1.5 ml400 mM Potassium ferrocyanide-20°C

Staining Solution 2

1.5 ml400 mM Potassium ferricyanide-20°C

Staining Solution 3

1.5 ml200 mM Magnesium chloride-20°C

10X PBS
100 ml				0.17 M KH2PO4
0.05 M Na2HPO4
1.5 M NaCl

RT or -20°C

10X Fixing Solution
17 ml				7% Formaldehyde
0.5% Glutaraldehyde
in 10X PBS-20°CX-gal

20 mg/ml X-gal in DMF

-20°C

8 ml

Additional materials required• Polypropylene or glass tube for Complete Staining Solution• 37°C incubator• Light microscope
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Protocols
PLEASE READ THE ENTIRE PROTOCOL BEFORE STARTING!
Warning: The 10X Fixing Solution contains formaldehyde and glutaraldehyde. Formaldehyde is poisonous and can be absorbed through the skin. Glutaraldehyde is corrosive and is a carcinogen that can also be absorbed through the skin. Potassium
ferricyanide and potassium ferrocyanide areharmful if swallowed, contacted with skin or inhaled.

Buffer Preparation
Dilute the 10X Fixing Solution and the 10X PBS in sterile H2O to a 1X final concentration.
Prepare fresh Complete Staining Solution using the chart below based on the number of samples
to be processed. Be sure to use a polypropylene plastic or glass tube.
Complete Staining Solution Preparation Chart
Note: Complete Staining Solution must be made fresh for each experiment.
Reagents

1 Plate (35 mm)

5 Plates (35 mm)

10 Plates (35 mm)

Staining Solution 120 µl

100 µl200 µlStaining Solution 220 µl

100 µl200 µlStaining Solution 320 µl

100 µl200 µl1X PBS1.84 ml9.20 ml18.4 ml

X-gal

100 µl500 µl

1 ml

Total

2 ml

10 ml

20 ml

Staining Protocol for a 35 mm Plate
1.

Aspirate the medium from the cells.

2.

Wash the cells with 3 ml 1X PBS.

3.

Add 2 ml 1X Fixing Solution to each well. Incubate at room temperature for 5-10 minutes.

4.

Wash the fixed cells twice with 2 ml 1X PBS.

5.

Add 2 ml Complete Staining Solution to each well.

6.

Incubate the cells at 37°C for 30 minutes to 2 hours, or as long as necessary to see blue staining of the cells.

7.

Check the cells under a microscope.

8.

Calculate the percentage of cells transfected with b-galactosidase.

9.

For long-term storage of the plates, remove the Complete Staining Solution and overlay thecells with 70% glycerol in PBS. Store at 4°C.
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2Appendix
Section A: Troubleshooting Guide
Problem/question

Possible CauseRecommendation

Cells do not stain blue

X-gal was not added to the Complete
Staining Solution.

Add X-gal to the plate(s).

Cells were not fixed correctly.

Fix cells for 10 minutes.

Unsuccessful transfection of DNA*.

Check transfection protocol and repeat
transfection using a control vector.

Construct is not expressing b-galactosidase*. Sequence the lacZ gene of your construct
to make sure it is in frame with an ATG.

All cells turned blue

Unsuccessful delivery of b-galactosidase
protein (when using Chariot™ Protein
Delivery Reagent).

Make sure b-galactosidase protein is stillactive. Consult the Troubleshooting Guideof the Chariot manual.

Improper washing of cells after fixative
treatment.

Glutaraldehyde will interfere with colordevelopment. Wash cells thoroughly.

Too much DNA was transfected*

Use less DNA in subsequent transfections.

Your cell line may be expressing endogenous Test this cell line using cells that have notb-galactosidase.
had b-galactosidase transfected/delivered.

Section B: Calculation of Efficiency
To calculate the percentage of cells expressing b-galactosidase, use the calculation below. This
calculation is also used for calculating successful delivery of the b-galactosidase protein using theChariot Protein Delivery Reagent.
Total number of blue cells x 100 = % transfection
Total number of cells

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3Technical Services
If you need assistance at any time, please call Active Motif Technical Service at one of thenumbers listed below.
Active Motif North America
1914 Palomar Oaks Way, Suite 150
Carlsbad, CA 92008
USAToll Free:877 222 9543
Telephone: 760 431 1263
Fax:
760 431 1351
E-mail:tech_service@activemotif.com
Active Motif Europe
104 Avenue Franklin Roosevelt
B-1330 Rixensart, Belgium
UK Free Phone:		
0800 169 31 47
France Free Phone:
0800 90 99 79
Germany Free Phone: 0800 181 99 10
Telephone: 		+32 (0)2 653 0001
Fax: 		
+32 (0)2 653 0050
E-mail: 		
eurotech@activemotif.com
Active Motif Japan
Azuma Bldg, 7th Floor
2-21 Ageba-Cho, Shinjuku-Ku
Tokyo, 162-0824, Japan
Telephone: +81 3 5225 3638
Fax:
+81 3 5261 8733
E-mail:japantech@activemotif.com
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