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Protein Binding

Binding Characteristics of Immobilon Membranes

PVDF is an inherently hydrophobic polymer and will not wet-out in aqueous solutions. In order for a PVDF membrane to be compatible with aqueous systems, it must first be wet in a 50% (v/v) or greater concentration of alcohol. Methanol, ethanol, and isopropanol are suitable to wet the membrane. Complete wetting is evident by a change in the membrane’s appearance from opaque to semi-transparent. The alcohol is then removed from the membrane by extensive rinsing in water, and the membrane can be directly equilibrated in transfer buffers.

Once the membrane is wet, protein binding can be achieved by simply bringing the protein into contact with the membrane. Because binding occurs throughout the depth of the membrane, the binding capacity is determined by the internal surface area of the pores (Mansfield, 1994). Immobilon-PSQ transfer membrane has approximately three times the internal surface area of Immobilon-P transfer membrane, resulting in higher adsorptive capacity (see the “Properties and applications of Immobilon PVDF transfer membranes” table earlier in this section). The values listed in the table represent upper limits for protein binding after saturation of the membrane surface in a non-denaturing buffer. In any given application, Immobilon-PSQ transfer membrane can be expected to bind more protein than Immobilon-P transfer membrane. However, the maximum binding that can be achieved will depend on the specific protocols employed, due to variations in the structural conformation of the proteins, the chemical nature of the buffers used, and the limitations of the methods used to apply the sample.

An example of the binding difference between Immobilon-P and Immobilon-PSQ transfer membranes is shown in the figure below, where protein samples were electrotransferred from a polyacrylamide gel.A fraction of the proteins passed through the Immobilon-P transfer membrane and were captured on a back-up membrane. In contrast, all of the proteins were bound to the Immobilon-PSQ coupon. In this case, the tighter pore structure and higher internal surface area of polymer facilitated complete adsorption of all of the transferred protein. However, immunodetection on Immobilon-PSQ transfer membrane can result in a higher background and can require more stringent washing conditions. Thus, the choice of membrane is dictated by the goal of the experiment: use Immobilon-P transfer membrane for high sensitivity detection of >20 kDa proteins, but switch to Immobilon-PSQ transfer membrane if smaller proteins are being analyzed or 100% protein capture is necessary for peptide sequencing.

Factors Affecting Protein Binding

At the molecular level, protein adsorption results, at least in part, from the interaction of hydrophobic amino acid side chains and hydrophobic domains with the polymer surface. Matsudaira (1987) observed an 80% decline in sequencing efficiency of small peptides after hydrophobic residues were cleaved, presumably due to washout of the peptide remnants. Also, in peptide digestions, it has been observed that peptides characterized as hydro-phobic often do not elute from the membrane as efficiently as more hydrophilic peptides (e.g., Iwamatsu, 1991; Fernandez et al.,1992) . McKeon and Lyman (1991) demonstrated that addition of Ca+2 ions to the transfer buffer enhanced the binding of calmodulin to Immobilon-P transfer membrane. Binding of the calcium causes formation of a hydrophobic pocket in the molecule’s structure.

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Prolonged Electrotransfer of Proteins using Immobilon-P and Immobilon-PSQ Transfer Membranes
Molecular weight standards (lanes 1,3,5,7) and calf liver lysate (lanes 2,4,6,8) were transferred to Immobilon-P or Immobilon-PSQ membranes by the tank transfer method and stained with Coomassie Blue. A sheet of Immobilon-PSQ transfer membrane was placed behind the primary membranes to capture proteins that passed through them. (Lanes 5 and 6 behind Immobilon-P; 7 and 8 behind Immobilon-PSQ.)

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Sample Blot
Detection of transferrin using Immobilon Western Chemiluminescent HRP Substrate on Immobilon-P membrane. Two-fold serial dilutions of transferrin were loaded and detected with goat anti-transferrin antibody (dilution 1:10,000) and HRP-conjugated rabbit anti-goat secondary antibody (dilution 1:100,000). Blot was exposed to the X-rayfilm for 1 minute.

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