AP Magenta 3 Component Membrane Substrate

AP Magenta 3 Component Membrane Substrate

Product No.: A327

[product_table name="All Top" skus="A327"]

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Product Type
Substrate and Assay Reagents
Applications
WB

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Product Size
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Product Details

Storage and Handling
Store at temperatures between 2-8ºC.
Expiration Date
Stable for a minimum of 18 months from the manufactured date when properly stored between 2-8ºC.

Description

Background
Leinco’s AP Magenta 3 Component Membrane Substrate is a precipitating substrate used with the enzyme alkaline phosphatase designed for membrane blotting procedures. The TAC Buffer coupled with a substitute, naphthol, is enzymatically hydrolyzed. In the presence of hexazotized substrate, a fine magenta precipitate is produced at the sites of alkaline phosphatase activity.
Components:
1. TAC Buffer (Modified Gormori’s Tris Azo-Coupling Buffer) - 4 x 100 ml or 1 x 1 L
2. Initiator (Hexa-azonium salt initiator) - 1 x 8 ml or 1 x 20 ml
3. Magenta Solution - 1 x 8 ml or 1 x 20 ml
Directions for Use
1. Add 2 drops, or 100 µl, of Magenta Solution into an appropriate sized test tube or collection vessel.
2. Add 2 drops, or 100 µl, of Initiator Solution to the 100 µl of Magenta Solution in step one. Gently mix and allow solution to sit for 3 to 5 minutes.
3. After the 3 to 5 minute incubation, add 5 ml of TAC Buffer and mix. USE IMMEDIATELY! (NOTE: Reagent is stable for up to one hour. Some color and turbidity may develop over this time frame. However, this does not affect product performance.)
4. Apply sufficient substrate solution to cover the membrane completely.
5. Incubate samples 10 minutes to 60 minutes, at room temperature, depending on individual preferences of staining intensity.

NOTE: Sites of enzymatic activity vary from pink to magenta depending in antigenicity and length of staining. Dilution of substrate is not recommended. To reduce the intensity of the reaction, it is recommended that antibodies or conjugates be diluted.
Troubleshooting
 
Problem Cause Solution
Too much background signal observed · BCIP/NBT substrate was left on the membrane too long · Decrease the amount of time the BCIP/NBT substrate is on the membrane
· Too much primary antibody used · Decrease the amount of primary antibody used and wash the membrane sufficiently after the primary antibody incubation
· Too much secondary antibody used · Decrease the amount of secondary antibody used
Nonspecific bands show up on the membrane · Too much primary antibody used · Decrease the amount of primary antibody used and wash the membrane sufficiently after the primary antibody incubation
· Too much secondary antibody used · Decrease the amount of secondary antibody used
Signal disappears from membrane · Membrane not stored correctly · Store the membrane dry in plastic bag in the dark
No signal is observed on the membrane · Low amounts of specific signal present · Expose the membrane to BCIP/NBT substrate for a longer period of time. Include positive control(s) during analysis
· Insufficient primary antibody used · Use more primary antibody
· Insufficient secondary antibody used · Use more secondary antibody
· Sample degraded · Add protease inhibitors to original sample before running a gel

Related Protocols

General Western Blot Protocol
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Products are for research use only. Not for use in diagnostic or therapeutic procedures.