A Model for Binding of DNA and Proteins to Transfer Membranes

This model can be expanded for protein and nucleic acid attachment to microporous membranes.  Among membranes used for molecular transfer applications, the highest binding levels are attained using hydrophobic PVDF membranes (13).  Water wettable nylon membranes contain strong hydrophobic components (7, 14) which can create microenvironments that encourage association with hydrophobic areas on biomolecules.

The importance of hydrophobic interactions can be shown by comparing nylon and PVDF membranes having different surface chemistries.  The presence of positively or negatively charged groups on the membrane that do not significantly affect the wettability of the membrane produce small changes in binding affinity.  The presence of hydroxyl groups increases the hydrophilicity of the membrane, stabilizes the layer of hydration around the membrane polymer, and significantly decreases the amount of binding.

Binding of biomolecules is measured as both affinity and avidity.  Each membrane type will have a characteristic affinity for biomolecules.  Adsorption is then dependent on the affinity and the input biomolecule concentration (10).

Bond strength (avidity) is also greatly influenced by the hydrophobicity of the membrane, and to a lesser extent, surface chemistry (14, 15).  A complex biomolecule has large numbers of potential sites for hydrophobic interaction so that very high bond strength can be achieved even though individual forces are weak.

A molecular model is shown, in which the layers of hydration surrounding the secondary structures of proteins or nucleic acids and membranes are squeezed out when they come in close contact.  The biomolecules then flatten out and increase the entropy of the system (10, 14), driving the interaction.  Desorption occurs when water re-enters the space between the molecule and the membrane.  The bond strength can be increased by desiccating the membrane-biomolecule complex.  This technique is commonly used as a fixation method for DNA detection (5).

Table 1: Membranes Used for Binding Studies

Test 1: Protein Binding by Immersion

A soak test was performed using 200 µg/mL goat IgG (Sigma, St. Louis, MO, USA) in PBS (20 mM phosphate, 0.15 M NaCl, pH 7) containing ¹²⁵I-goat IgG tracer (NEN, Boston, MA, USA). 13 mm disks of each membrane type were soaked in 2 mL per disk of the protein solution for one hour with agitation.  Disks were washed three times, 5 minutes per wash, with PBS to remove loosely bound protein.  Disks were then counted.  After counting, the disks were washed twice with 2 mL per disc of 1% SDS, 2 M urea, 15 minutes per wash, to remove remaining protein that is not tightly associated with the membrane.  After washing, disks were rinsed with water and counted again.

Test 2: Protein Binding by Perfusion

A PBS solution containing goat IgG and ¹²⁵I-goat IgG tracer were perfused through 13 mm disks of membrane.  Total influent protein was varied from 1 µg/cm² to 30,000 µg/cm².  After perfusion, disks were blotted on a filter paper and counted.

Test 3: DNA Binding by Immersion

13 mm disks of each membrane type were soaked in 2 mL per disc of 2X SSC containing Lambda Hind III DNA digest (BRL Life Technologies, Bethesda, MD, USA) and ³²P labeled Lambda Hind III DNA (prepared by nick translation using reagents from BRL).  DNA concentration was varied between 30 ng/cm² and 10 µg/cm².  Disks were soaked in DNA solution for 60 minutes.  Disks were then rinsed in 2X SSC, blotted on filter paper and counted.

Test 4: DNA Dot Blot

Lambda Hind III DNA digest diluted in 2X SSC was spotted on membrane cards in concentrations from 100 pg to 0.3 pg per spot.  Membranes were fixed as follows: Biodyne A, Biodyne Plus, Biodyne C and LoProdyne LP membranes were baked at 80 °C for 30 minutes and then exposed to UV light (Mineralight R52) for 3 minutes.  Biodyne B membranes were baked at 80 °C for 30 minutes.  DNA was detected on the membrane using the Boehringer Mannheim GeniusTM kit (Boehringer Mannheim, Indianapolis, IN, USA) , with digoxigenin labeled Lambda Hind III probe and BCIP/NBT color forming substrate.

As shown for solid surfaces, there is a general correlation between hydrophobicity of a membrane and the amount of binding (Figure 1).  The hydrophobic PVDF FluoroTrans membrane consistently demonstrated the highest level of binding.  On hydrophilic nylon membranes, the presence of charged groups had only a small effect: Biodyne B membrane (positive surface charge) bound slightly more protein than Biodyne C membrane (negative surface charge).

The hydroxyl chemistry on the LoProdyne LP membrane increases the hydrophilicity of the membrane and stabilizes the layer of hydration around the nylon molecules.  This limits opportunity for hydrophobic interaction and results in very low protein adsorption.

Protein Binding Test

Figure 1:  Protein binding to different membrane types. Membrane disks were soaked in buffer containing IgG and ¹²⁵ I labeled IgG tracer.  Disks were rinsed with PBS and counted.  To determine bond strength, disks were then washed with 1% SDS, 2M urea, rinsed and counted again.

Protein Binding at Different Challenge Levels

Soak tests with varying amounts of IgG protein showed that total binding to the membrane increased continuously with increasing input concentration, although the fraction of protein bound slowly decreases (Figure 2). 

The highest amount of protein bound was greater than 30,000ug/cm².  Proteins will bind to flattened proteins already on the membrane surface, forming layers (10). These protein-protein interactions make it impossible to define a “binding capacity” for a membrane.  The amount binding will be determined by the amount of protein applied to the membrane.

Protein Binding to Nylon Membrane

Figure 2:   Protein binding test.  Buffered saline solution containing IgG protein and ¹²⁵I labeled IgG tracer was perfused through nylon disks at concentrations between 3µg/cm² and 30mg/cm².  After perfusion, disks were blotted and counted.

Binding of DNA Due to Hydrophobic Interaction

An immersion binding test using different concentrations of  ³²P labeled Lambda Hind III DNA is shown in Figure 3.  As with proteins,  the hydrophobic FluoroTrans membrane resulted in the highest binding at all input concentrations.  Biodyne B membrane (positive surface charge) yielded higher binding than Biodyne C membrane (negative surface charge).  The presence of hydroxyl groups on LoProdyne LP membrane resulted in very low binding of DNA, again highlighting the importance of hydrophobic interactions.

Figure 3: DNA binding to different membrane types.  13mm disks of each type were soaked for 60 minutes in 2 mL buffer per disk containing Lambda Hind III DNA and ³²P labeled tracer.  After soaking, disks were blotted dry and counted.

Surface Chemistry and DNA Binding

A dot blot test was performed to compare the different membranes in a typical DNA detection system.  A test performed with the Boehringer Mannheim Genius™ system is shown in Figure 4.

As predicted by the DNA binding test, Biodyne C membrane achieved good sensitivity despite the negative charge present both the membrane and the DNA molecule.  FluoroTrans membrane typically yields performance as good or better than the nylon membranes (not shown). 

Similar DNA sensitivity has been obtained with various DNA detection systems using Biodyne A, Biodyne B, Biodyne Plus and FluoroTrans membranes.

Figure 4:  DNA dot blot test.  Dilutions of Lambda Hind III DNA were applied to membranes as 1.5 µl spots.  Membranes were air dried and fixed by baking (Biodyne B membrane) or UV exposure (all other membranes).  Membranes were hybridized according to Boehringer Mannheim recommendations, using Lambda Hind III probe labeled with digoxigenin.  After hybridization, signal on the membranes was developed with BCIP/NBT substrate.

An examination of the molecular structure of nylon and PVDF membranes shows how hydrophobic bonds can form in membranes that are wet with water:

Nylon Membranes

Pall nylon membranes are cast from nylon 6,6:

The polymer structure is mostly non-polar with terminal amino and carboxyl groups.  When formed into a membrane (7), the molecule can exhibit a structure like the one shown in Figure 5.  The hydrophobic regions fold away from the surface of the pores so that the terminal polar groups are exposed.  In this manner, a hydrophilic membrane can be formed from hydrophobic molecules.

Figure 5: Probable conformation of nylon 6,6 polymner cast into a water-wettable membrane.

PVDF Membranes

Unmodified polyvinylidene fluoride membrane is hydrophobic and contains no charged groups for electrostatic interaction.  Before use, air in the pore structure must be displaced using a low surface tension liquid (methyl alcohol), which can then be exchanged to water or buffer.

Polyvinylidene Fluoride:

A Molecular Model

Biomolecules having secondary and tertiary structure are transported to the membrane surface by diffusion.  When a structured molecule makes contact with the membrane, interactions occur between hydrophobic areas on the membrane and hydrophobic areas on the biomolecule (Figure 6).  As this happens, the water layers surrounding the biomolecule and the membrane are squeezed out.  As this occurs, the biomolecule loses structure.  Large hydrophobic domains on both the biomolecule and the membrane can result in very strong associations.

This model is easier to visualize for proteins, which normally exist in globular shapes and contain complex combinations of charged groups, than for nucleic acids. 

Nucleic acids have a regular structure and a negative charge at neutral pH.  Despite this, nucleic acids bind to membranes in the same manner as proteins. There is enough secondary structure in the nucleic acid to favor hydrophobic binding when the molecules are brought into close contact with the membrane surface.

Figure 6: Association between protein and nylon membrane.

1. Transport of the biomolecules to the membrane surface by diffusion; 2. Alignment of hydrophobic domains on the biomolecule and the membrane; 3. Penetration of hydrophobic regions on the biomolecule into hydrophobic regions in the membrane; 4. Elimination of layers of hydration surrounding these regions on both the membrane and biomolecule.

The model explains the high affinity of hydrophobic PVDF membranes for proteins and nucleic acids, as well as the high bond strength.  The model also predicts low binding to hydroxyl modified membranes, and how biomolecules can be forced to attach to intrinsically low binding membranes by removing water.

In this model, the term binding capacity is replaced with binding affinity.  A membrane will have an affinity for biomolecules dependent on its hydrophobic components and, to a much lesser extent, surface chemistry.  These factors are predictive of how “sticky” a membrane is, and how it is likely to perform in biological applications.

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