Binding Assays with Membranes

The purpose of the binding assay is to determine the amoimt of bound ligand. For this, the assay separates the bound from the free ligand. For membranes this is easy, because the physical fference is large between membrane vesicles and the radioactive ligand (from simple molecule to protein). 

In the centrifugation assay the membranes settle with the ligand, which is bound to them. The free ligand remains in the supernatant. 

The experimenter resuspends soft pellets by vortexing. If the pellets stick to the wall of the Eppendorf vessel, he resuspends by repeatedly pulling pellet and binding buffer into a Gilson pipette or scrapes the tip of the vessel a few times along a steel net (e.g., autoclave basket). For a binding solution without membranes, the careful researcher makes sure that her ligand does not bind to the Eppendorf vessels themselves. 

Immediately after the supernatant is completely siphoned off, the filters are washed once or twice with cold washing buffer. The washing buffer can be identical to the binding buffer, but if possible you should leave out the expensive components of the binding buffer. With H-marked ligands, the filters have to equilibrate with the scintillator before the counter is applied. Equilibrating takes hours. Shaking of the samples accelerates it. The filtration devices from Millipore and Hoefer are expensive ( 1,000 to 1,500). An alternative is to build similar devices. 

Adsorption on glass fiber filters is simple, inexpensive, and popular. The large internal surface of the filters adsorbs membrane vesicles via weak electrostatic and hydrophobic interactions. Bigger vesicles (mitochondriae, synaptosomes) are also filtered mechanically (pore size of glass fiber filters 1 to 3 pm 0 mitochondriae 1 pm). Molecules in solution largely 

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Experimental agents were evaluated in a receptor binding assay with 3H-nociceptin/orphanin FQ with membranes of recombinant CHO-ORLj cells according to the method of Ardati (1). Testing results are provided in Table 2. 

Wiltschi B, Knoll W, Sinner E-K (2006) Binding assays with artificial tethered membranes using surface plasmon resonance. Methods 39 134-146... 

In 1992, Kaminski and colleagues (Kaminski et al. 1992) demonstrated through an equilibrium binding assay that membranes from mouse spleen had specific binding sites for cannabinoids. In addition, using specific primers for the cannabinoid receptor identified in brain, they amplified from splenic RNA using RNA transcriptase-polymerase chain reaction (RT-PCR), an 854-kb product that hybridized with brain cannabinoid receptor cDNA. These studies demonstrated that... 

This interpretation is supported by data from radioligand studies 8). According to saturation binding assays with [ Hjthiamethoxam and competition assays with [ Hjimidacloprid, all the compounds under discussion exhibit about the same affinity to aphid membranes. The data, given as Kq and /Cj values, respectively, for M persicae are (5) ... 

Equilibrium binding assays with [ HJryanodine (Perkin Elmer, spec, activity 2.072 TBq mmof ) and [ HJflubendiamide (in-house synthesis, 905.6 GBq mmof ) were performed on microsomal membrane preparations from Heliothis virescens flight muscles as described previously (16). 

Enzyme Immunosensors. Enzyme immunosensors are enzyme immunoassays coupled with electrochemical sensors. These sensors (qv) require multiple steps for analyte determination, and either sandwich assays or competitive binding assays maybe used. Both of these assays use antibodies for the analyte of interest attached to a membrane on the surface of an electrochemical sensor. In the sandwich assay type, the membrane-bound antibody binds the sample antigen, which in turn binds another antibody that is enzyme-labeled. This immunosensor is then placed in a solution containing the substrate for the labeling enzyme and the rate of product formation is measured electrochemically. The rate of the reaction is proportional to the amount of bound enzyme and thus to the amount of the analyte antigen. The sandwich assay can be used only with antigens capable of binding two different antibodies simultaneously (53). 

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