NEL602, NEL602A, Western Lightning - Certificate of Analysis

Technical Data
Certificate of Analysis
PerkinElmer Life Sciences, Inc.
549 Albany Street
Boston, MA 02118
Caution: For Laboratory Use. A research chemical for research purposes only.
WESTERN LIGHTNING™
CDP-Star® Chemiluminescence Reagent (Ready-to-Use)
125 mL for 2500 cm-12,500 cm2, 50 mL for 1000 cm-5,000 cm2
Catalog Number:
Shipping Conditions:
Storage Conditions:
NEL602, NEL602A
Ambient Temperature
4°C
Product Description
Western Lightning™ CDP-Star Chemiluminescence Reagent (Ready-to-Use) contains the chemiluminescence
substrate CDP-Star (MW=495.2) for the detection of alkaline phosphatase. The reagent, prepared in a
proprietary buffer, is ready to use and does not require any further dilution.
Introduction
CDP-Star is a non-radioactive light emitting reagent used to detect the enzyme alkaline phosphatase immobilized
on a membrane. Maximum light emission occurs two hours after substrate incubation with continuous light output
for a minimum of 24 hours (See Figure 1).
When used in Western Blotting applications, picogram quantities of protein can be detected. Film or KODAK
Image Station exposure to achieve this level of sensitivity can be 1 second to 10 minutes.
Light Emission Kinetics of CDP- Star® on Nylon Membrane
Figure 1
Intended Use
Intended use of this product is the detection of alkaline phosphatase.
Performance Considerations
1.
This reagent has been formulated and quality controlled specifically to detect alkaline phosphatase antibody or
streptavidin conjugates immobilized onto membranes.
2.
Western Lightning™ CDP-Star Chemiluminescence Reagent (Ready-to-Use) is applicable to PVDF membranes
such as PolyScreen® PVDF Transfer Membrane and also nylon membranes such as GeneScreen™ and
GeneScreen Plus®.
3.
KODAK X-OMAT Blue Film or the KODAK Image Station, ideal for chemiluminescence CDP-Star detection, are
recommended for best results.
Protocol for Use
The following is a protocol for using CDP-Star in a Western Blot experiment. The PolyScreen PVDF Transfer Membrane
product insert contains detailed protocols for membrane wetting and Western Blotting.
1.
If PolyScreen has dried after completion of Western Blotting procedure it must be re-wet with
95% ethanol for at least one minute. Rinse the membrane in distilled water to wash off the alcohol for
2-3 minutes.
2.
Block the membrane in Blocking Buffer [PBST (0.05M phosphate, 0.154M NaCI, 0.05% TWEEN 20)] for 1 hour at
room temperature. To prepare blocking buffer add blocking reagent slowly to the PBST while stirring. Continue
stirring and heat the solution slowly to 60°C until all of the blocking reagent is dissolved. The resulting solution
will be opaque. Cool to room temperature before use. Alternatively, the membrane may be blocked in 5% nonfat dry milk in PBST.
3.
Dilute primary antibody in Blocking Buffer and incubate for at least 1 hour at room temperature.
4.
Wash membrane 2 X 5 minutes in Blocking Buffer at room temperature.
5.
Dilute secondary antibody in Blocking Buffer and incubate for at least 1 hour at room temperature.
6.
Wash membrane 3 X 5 minutes in Blocking Buffer at room temperature.
7.
Mark the membrane to distinguish the side with the immobilized alkaline phosphatase and wash for
5 minutes in 0.1 M Tris (pH 9.5).
8.
Place the membrane in a clean dish or hybridization bag.
Carefully add CDP-Star to membranes
(0.01 mL/cm2 -0.05 mL/ cm2). Incubate for 5 min at room temperature with agitation. The blot should be covered
with solution during this incubation.
9.
Remove the blot from the substrate and gently blot off any excess reagent onto any filter paper or paper towels.
Do not allow the membranes to dry.
10.
Place the membranes, protein side up, between plastic sheet protectors (Boise Cascade Catalog Number L2A8112) or in plastic wrap and expose initially to KODAK X-OMAT Blue film or the KODAK Image Station for 5-10
minutes. Re-exposures can be done until optimal signal to noise ratios are achieved as chemiluminescence
signal from CDP-Star continues for at least 24 hours.
PC2405-0202
Stability
The reagent is stable for at least one year from the date of manufacture when stored properly at 4°C.
Storage
Western Lightning™ CDP-Star Chemiluminescence Reagent (Ready-to-Use) is shipped ambient and should be placed at
4°C upon arrival for maximum stability.
Safety Considerations
Caution: For Laboratory Use. A Research Chemical for Research Purposes Only.
Warning: Harmful by Contact, Ingestion, Inhalation. Irritating to Eyes, Skin, & Respiratory Tract; Slightly Toxic.
Target Organs: Eyes, Upper Respiratory Passages, Skin
Trademarks
CDP-Star is a registered trademark of Applied Biosystems, Inc. CDP-Star is protected under one or more of U.S. Patents
5,326,882; 4,931,569; 5,582,980; 5,851,771; 5,538,847; 5,145,772; 4,978,614.
PC2405-0202
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