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Index / Trevigen / Cell Permeabilization Solution / Product Detail : 4674-250-01 Cell Permeabilization Solution
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Dec
10th

#4674-250-01 Cell Permeabilization Solution

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  Price : 361   EUR
410   USD
280   GBP
1518   Zloty
48381   JPY
2788   NOK
2987   SEK
408   CHF

Product name : Cell Permeabilization Solution

Catalog number : 4674-250-01

Quantity: 250 µl

Availability: Yes

Supplier name : Trevigen

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More Details about

Description: Cell Permeabilization Solution allows for the efficient transfer of Fluorescein-conjugated NAD across cellular membranes. The solution contains a selective cell permeable peptide to deliver Trevigen’s Fluorescein-NAD (cat#4673-500-01) from the outside to the inside of intact cells.
Physical State: Peptide provided as solution in deionized water.
Storage: Store at -20°C.
Applications:
¨ In-cell activity measurements of NAD-requiring enzymes.
¨ In-cell Assays to identify inhibitors of activators of NAD-requiring enzymes.


In Cell Assay:
1. Culture cells in media as recommended by supplier, and allow at least two passages prior to assay. Adherent cells may also be cultured in chamber slides.
2. Treat cells as desired; it is recommended to treat one sample with 2 mM 3-aminobenzamide as a control. Some treatments may require cells be incubated in serumfree medium prior to treatment. Allow at least 30 minutes to prepare reaction cocktail.
3. Prepare 400 μl of Fluorescein-NAD reaction cocktail per 1.0 x 106 cells. Store reaction cocktail in the dark for at least 10 minutes prior to adding cells.


Reaction Cocktail for 25 μM Fluorescein-NAD reaction per 400 μl:
Sterile, deionized water 296 μl
20X PARP Buffer 20 μl
100 mM DTT 4 μl
Cell Permeabilization Solution 40 μl
250 μM NAD-Fluorescein 40 μl
400 μl
Note: Reaction Mix can also be prepared by first mixing one part Fluorescein-NAD with one part Cell Permeabilization Solution and incubate in the dark for at least 10 minutes prior to the addition of the remaining ingredients.

4. Wash cells twice with PBS, and add 200 μl of Reaction Cocktail per 5.0 x 105 cells. Incubate cells at 37°C for one hour.
5. Discard Reaction Cocktail, and incubate cells in chilled 95% Ethanol (-20°C) for 10 minutes to fix cells.
6. Discard 95% ethanol, and incubate cells in chilled 10% TCA (4 °C) for 15 minutes to inactivate PAR glycosylases.
7. Wash cells with PBS, mount with fluorescent mounting media, and view under fluorescent microscope.

 

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