Parallel Artificial Membrane Permeability Assay-BBB Kit

Protocol SDS

Application

• For quantitative determination of blood brain barrier (BBB) membrane permeability of test compounds.

Key Features

• Convenient. Includes all necessary equipment to run a PAMPA plate.
• Simple and low-cost. Procedure is easy to follow and more affordable than cell-based permeability assays.
• High-throughput. Can be readily automated as a high-throughput 96-well plate assay for thousands of samples per day.

Method

• PAMPA

Samples

• Chemical compounds

Procedure

• Assay takes 20 hr, hands-on time 1 hr

Size

• 96 tests

Shelf Life

• 12 months

More Details

MEMBRANE PERMEABILITY is an important characteristic to determine for evaluating compounds as potential drug candidates. Drugs often need to cross cell membranes in order to reach their target of action and this makes a compound’s ability to passively cross these membranes an important characteristic to evaluate. The Blood Brain Barrier (BBB) is made of brain endothelial cells with tight junctions. Rapid and early screening of compounds for BBB penetration is highly desirable for drug discovery. Permeability can be evaluated by cell-based methods; however, these methods are often expensive and time-consuming. Parallel Artificial Permeability Assays (PAMPA) offer researchers a quick, inexpensive method of evaluating the permeability of test compounds. Our PMBBB-096 kit is designed to aid in evaluating BBB permeability. BioAssay Systems’ PMBBB Kit provides all the necessary components to run a Parallel Artificial Permeability Assay for Blood Brain Barrier studies.

What are the Permeability Controls?

The High and Low Controls are 10 mM stock solutions of Promazine Hydrochloride and Diclofenac, respectively, in DMSO.

What other materials are required to run the assay?

Pipetting devices, DMSO, PBS, UV Plates (Cat # P96UV), and an absorbance plate reader capable of absorbance spectra.

What is the membrane of the donor plate?

The donor plate membrane is a Hydrophobic PVDF membrane with a pore size of 0.45 µm.

How long are the solubilized BBB lipids stable for?

Once solubilized, the lipids should be stored at -20C and used within 6 days.

My test compounds aren?t soluble at 500 µM in PBS. Can I use higher concentrations of DMSO in the PAMPA assay?

We recommend against using higher concentrations of DMSO in the PAMPA assay. The spectrophotometric background in the Acceptor solution will increase with higher DMSO concentrations, and the higher concentration of DMSO may alter the membrane and affect the result.

If your compound(s) is/are insoluble in PBS with 5% DMSO, clarify the solution by centrifugation at 13,000xg for 10 minutes then use the supernatant for the Donor solution. The Donor solution is used in place of “500 µM Test Compound”. Since some of the compound was centrifuged out, the new concentration is unknown, so it is instead referred to as Donor Solution.
For the Equilibrium Standard, mix 80 µL of the new Donor Solution with 120 µL of PBS.

Didn’t the kit used to come with three permeability controls?

Yes, the kit used to utilize Clonidine as a medium permeability control, but we discovered that this control was subject to oxidative degradation, so we have omitted from the current version of the kit. We continue to look for a replacement medium permeability control, but its absence from the kit doesn’t undercut the performance of the kit.

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If you or your labs do not have the equipment or scientists necessary to run this assay, BioAssay Systems can perform the service for you.

– Fast turnaround
– Quality data
– Low cost

Please email or call 1-510-782-9988 x 2 to discuss your projects.