D-Tube™ Dialyzers Provide High Protein Recovery

Introduction

Dialysis is commonly used to separate sample components by size. Separation is achieved by selective diffusion across a porous, semi-permeable membrane, which allows permeation of small molecules (e.g., solvents, salts, and small metabolites) while blocking the passage of larger molecules. Traditional dialysis tubing can be awkward to handle; leakage and sample loss can occur if tube ends are not sealed effectively.

As an alternative, Novagen® D-Tube™ Dialyzers are easy-to-use devices in a screw-cap tube format. D-Tube™ Dialyzers bear dual dialysis membrane windows for buffer exchange. These disposable tubes are easy to handle for sample manipulation, requiring neither syringes nor microcentrifugation. The sample is added and removed using a standard laboratory pipette. D-Tube™ Dialyzers are available in five different versions to accommodate a range of sample volumes: Mini (10–250 µL, Midi (50–800 µL), Maxi (0.5–3 mL), Mega (3–20 mL), and in D-Tube96™ format for 96-well plate applications. The D-Tube™ Dialyzer Maxi kits are provided with two caps to allow easy adjustment of sample volumes between 0.5–2 mL and 2–3 mL (Figure 1). Similarly, the maximum volume capacity of D-Tube™ Dialyzer Mega devices is determined by cap size, with tubes and corresponding caps available for 10-, 15-, or 20-mL maximum capacities. The D-Tube™ Dialyzers are available in a variety of membrane molecular weight cut-offs. The design of the Mini, Midi, and Maxi tubes also permits their use for electroelution of proteins or nucleic acids from acrylamide or agarose gels.

 

D-Tube Dialyzer Maxis are provided with caps designed to accomodate a maximum internal volume of 3-ml (left panel) and 2-ml (right).

Figure 1. D-Tube™ Dialyzer Maxis are provided with caps designed to accomodate a maximum internal volume of 3-mL (left panel) and 2-mL (right).

Testing Protein Binding of the D-Tube™ Dialyzer Plastic

Unlike dialysis with traditional tubing, samples in D-Tube™ Dialyzers are in contact with the plastic of the lid and wall supports (Figure 1). To test the propensity of this plastic to bind proteins, we dialyzed solutions of representative acidic, neutral, or basic proteins and compared sample protein concentrations before and after dialysis. We chose low protein concentrations, as this is where loss due to non-specifc binding to plastic is most problematic.

 

Methods

We selected three proteins to test for binding to the D-Tube™ dialyzer plastic: soybean trypsin inhibitor (SBTI; isoelectric point (pI) 4.6), a recombinant form of the 3C protease from human rhinovirus type 14 (HRV 3C; pI 7.3), and bovine pancreatic ribonuclease A (RNase A; pI 9.4). Stock solutions of each protein were prepared in phosphate buffer (100 mM NaCl, 50 mM sodium phosphate, pH 8.0) at concentrations ranging from 35 to 70 µg/mL. We selected these concentrations based on the lower sensitivity limit of the BCA assay, to ensure accurate and reliable measurements even in the event of substantial protein loss after dialysis.

For each test protein, we performed four independent dialysis experiments (Figure 2). Four 2 mL samples of protein stock were added to D-Tube™ Dialyzer Maxi units (MWCO 3.5 kDa;), which were closed with the 2-mL caps. The D-Tube™ devices were assembled in a floating rack and placed in a beaker containing 1 L phosphate buffer at the same ionic strength used for protein stock preparation. Dialysis was performed overnight at 4 ˚C with gentle stirring. Concentrations of pre-dialysis protein stock solutions and dialyzed samples were measured using the BCA Protein Assay Kit. All measurements were performed in quadruplicate.

 

Protein concentrations before and after D-Tube dialysis

Figure 2. Protein concentrations before and after D-Tube™ dialysis. The blue bars show the concentrations of the stock protein solutions before dialysis; the tinted green bars represent the four independent dialysis experiments for each protein. The average of four BCA assay measurements for each sample is shown ±1 standard deviation.

Results and Discussion

At the relatively low protein concentrations tested (35–70 µg/mL), no significant protein loss occurred (Figure 2). These results demonstrate that, over a range of net protein charge, protein binding to the plastic of the D-Tube™ Dialyzer Devices is insignificant. Because the ratio of plastic surface area to sample volume is smaller for the D-Tube™ Mega Dialyzer devices, this conclusion is valid for larger-volume dialysis applications as well. D-Tube™ Dialyzers are thus suitable for sensitive applications that require high protein recovery after dialysis.

Materials