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ReadiUse™ probenecid *25 mM stabilized aqueous solution*

Product Information
Unit Size: Cat No: Price (USD): Qty:
10x10 mL 20062 $145
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Telephone: 1-800-990-8053
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Chemical structure for ReadiUse™ probenecid *25 mM stabilized aqueous solution*.
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Overview

Ex/Em (nm) None/None
MW 307.34
CAS # N/A
Solvent N/A
Storage F/D/L
Category GPCR
Calcium GPCR Assays
Related Cell Signaling
Biochemical Assays
Probenecid is an inhibitor of organic-anion transporters located in cell membranes. These transporters often extrude fluorescent indicators from cells, and therefore contribute to poor dye retention. This phenomenon usually causes high background in the assays that require the good retention of the dye indicators inside cells. The use of probenecid to inhibit the transporter activity, and thus to reduce leakage of the intracellular dye indicators is a common method for reducing fluorescence background of calcium assays. The commonly used free acid form of probenecid requires the use of 1 M NaOH to dissolve it due to its poor water solubility in neutral water. AAT Bioquest offer the convenient Readiuse™ water-soluble and heat-stable probenecid in the format of a powder, solution or tablet. They are convenient to use and are as effective as the free acid form at the same concentration. Each vial is diluted with 90 mL buffer to make 2.5 mM final concentration of probenecid for your assays.




Protocol


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This protocol only provides a guideline, and should be modified according to your specific needs.

1         Thaw 1 bottle of Cat. # 20062 at room temperature before use.

2         Make 2.5 mM ReadiUse™ probenecid buffer: Add 1 ml of ReadiUse™ probenecid 25 mM stabilized aqueous solution to 9 ml of HHBS (1X Hank’s with 20 mM Hepes buffer, pH 7.0), and mix them well.

Note1: 10 mL of 2.5 mM Readiuse™ probenecid buffer is enough for 1 plate. Aliquot and store the unused Readiuse™ probenecid 25 mM stabilized aqueous solution at < -20 oC, protected from light. Avoid repeated freeze-thaw cycles.

Note2: 2.5 mM Readiuse™ probenecid buffer contains 0.02% Pluronic® F-127, so it is not necessary to add extra Pluronic® F-127in the dye-loading solution.

3         Make dye-loading solution for one cell plate: Add DMSO reconstituted fluorescent calcium dyes (such as Indo-1 AM, Fura-2 AM, Fluo-3 AM, Fluo-4 AM, Fluo-8 AM, Rhod-2 AM and Rhod-4 AM) into 2.5 mM ReadiUse™ probenecid buffer, and mix them well. The working solution is stable for at least 2 hours at room temperature.






References & Citations

Teleocidin A2 inhibits human proteinase-activated receptor 2 signaling in tumor cells
Authors: Sonja Stahn, Lisa Thelen, Ina-Maria Albrecht, Jens Bitzer, Thomas Henkel, Nicole Elisabeth Teusch
Journal: Pharmacology Research & Perspectives (2016)

Glutathione adducts on sarcoplasmic/endoplasmic reticulum Ca2+ ATPase Cys-674 regulate endothelial cell calcium stores and angiogenic function as well as promote ischemic blood flow recovery
Authors: Melissa D Thompson, Yu Mei, Robert M Weisbrod, Marcy Silver, Praphulla C Shukla, Victoria M Bolotina, Richard A Cohen, Xiaoyong Tong
Journal: Journal of Biological Chemistry (2014): 19907--19916






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