July 1, 2016 (Vol. 36, No. 13)

Peter Rezk Global Market Manager Wheaton

AntiBIND: Ultimate Recovery of Your Valuable Proteins at Low Concentrations

Microplates are the workhorses of the lab. They are used in almost every application, from sample collection and preparations to measuring the faint signal of a low abundant biomarker in a blood sample. In this tutorial, we will examine the suitability of using AntiBIND family of microplates in protein research applications such as protein storage, high throughput protein discovery, biochemical analysis via chromatographically application LC/MS.

Starting with the Right Raw Materials

When it comes to lab consumables such as microplates, micro tubes, and other disposable containers, the plastic of choice is polypropylene (PP). PP is rugged and resistant to many chemicals, acids, and bases with a melting point of anywhere from 130 °C (266 °F) up to 171 °C (340 °F). An essential aspect of manufacturing these consumables is the selection of the right raw materials. One of the best suited PP grade for lab consumables is homopolymer PP grade using metallocene catalysts. These resins have improved optical and processing characteristics, combined with exceptional purity, and minimal additives that can be leached into the solution. Unfortunately, many companies use injection molding grades of PP with high impurities and add to the resin other extractables additives to increase manufacturing output at lower costs. These leachate compounds can be grouped into groups such as mold releasing agents, polymer flow improvers, surface coating, and or other materials that allow for a modification in the plastic outer properties. The leaching of these additives may interfere with solute and or proteins in solution jeopardizing sample integrity and biochemical results. The negative impact of this leaching phenomenon is well reported and observed in long term sample storage applications, solutions containing solvents such as methanol, DMSO, or detergents such as EDTA and or high/low pH solutions.

We tested AntiBIND as well as five other treated and untreated microplates available in the market to understand the extractions profile of these plates and the different challenges they may pose to the researcher. The plates were cryogenically ground in a freezer mill to produce a high surface area and then extracted with chloroform for two hours. After filtration, extracts were analyzed by GC-MS and LC-MS. Additional extraction experiments conducted via regular incubation with isopropanol, and methanol were performed to determine extractable profile.

Whether extracting with chloroform or isopropanol or evaluating via GC-MS or LC-MS, AntiBIND polypropylene (PP) plates consistently showed superior extraction performance when compared to five other competitive grades of treated and untreated PP plates and specialty plates. Additionally, low protein binding plates offered in the market leached the hydrophilic moiety in the presence of these solvents exposing the hydrophobic PP areas. (Check www.wheaton.com/AntiBIND for detailed experiments on extractable and leachable.)

Figure 1. AntiBIND 96 deep well plate, volume 0.5 mL with conical well design

Using Advanced Plasma Technology to Alter the PP Properties from Hydrophobic to Hydrophilic to Minimize Protein-Wall Binding without the Addition of Leachables

Polypropylene is a hydrophobic material made up of the non-polar monomer containing CH, CH2, and CH3 chemical groups. Most studies have shown that the adsorption process, or non-specific protein binding, is reduced by decreasing the hydrophobicity of the sorbent surface. While this can be done by different mechanisms such as applying a hydrophilic coating (PEG-modified surfaces and siliconization for glass) or incorporating hydrophilic compounds, these treatments are unstable and have the tendency to easily leach into solution exposing the hydrophobic parts of the plate. This was the case with the leading brand of low protein binding plates in the presence of commonly used organic solvents.

AntiBIND employs the use of Advanced Plasma Technology. Plasma surface activation is the process by which surface polymer functional groups are replaced with different atoms from ions in the plasma to increase surface energy. Surface exposure to energetic species breaks down the polymer at the surface, creating free radicals. Free radicals quickly react with the material itself because they are unstable, which allows for the forming of stable covalent bonds.

Proprietary mixture of plasmas made up of oxygen and water vapor were applied to the surface of the polypropylene modifying the surface of polypropylene with predominantly hydrogen-bond-acceptor uncharged polar groups. This treatment results in the formation of a variety of oxygen- and nitrogen- containing polar groups including C-O-C bonds, C=O and O-C=O, and C-N. The resultant is an increased surface hydrophilicity as measured by the decrease in water contact angle from 82° for the untreated polypropylene microplates to 60° for the AntiBIND microplate.

Significant Reduction in Protein Loss

Since microplates are used in a plethora of applications that may include 1000s of different proteins, we devised various experiments to best capture these different factors in our protein adsorption and protein recovery experiments.

To measure the amount of protein adsorbed to the plate, we measured the protein recovery rates of different fluorescently tagged proteins after different incubation times in the plates and at varied concentrations. The various sets of experiments were performed on the AntiBIND microplate, the best in class low protein binding plates, and standard high-quality PP plates. The results show a significant decrease in the adsorption (non-specific protein binding) and consequently a signification increase in protein recovery. The AntiBind plate clearly demonstrates a significant competitive advantage over the standard and low protein binding microplates commercially available today, particularly when the application deals with low protein concentrations. The nonspecific proteins binding process happened immediately after the addition of the proteins with most of the binding occurring within the first 4hrs. This trend was seen in all of the proteins that were tested and was very apparent at low concentrations.

Figure 2. AntiBIND plates do not show extractables species, whereas the competitive plates do leach components that interfere with detection and analysis. The stripping of the low binding capability of the competitors’ plates will decrease its low binding capability and allow for greater protein adsorption to the wall of the plate.

Robustness of Use and Compatibility with Automation for Chromatography Applications

The American National Standards Institute, Inc. (ANSI) and the Society for Laboratory Automation and Screening (SLAS) have defined the dimensional standards of microplates to ensure they work with most automated systems. The AntiBIND family of plates, 96 square well (0.5mL, 1.0 mL and 2.0 mL) and the 384 well (120 µL), are designed and manufactured according to these guidelines enabling compatibility with all automated systems. Moreover, they are produced in class 7 clean room under Dnase/Rnase free environment. The conical bottom of the 0.5mL and the round bottom of the (1.0 mL and 2.0 mL) allows for easy pipette access and maximum sample recovery. Additionally, the 96-well plates have a raised rim that forms a tight seal, when using a sealing mat, to minimize sample loss due to evaporation and cross-talks between wells.

In conclusion, even though microplates manufactured by different companies may look the same, there are major differences among them. The purity of the PP resin will have a tremendous effect on the integrity of the sample and the bioanalytical results. It is important to use the right raw materials (homodimer PP resin manufactured via metallocene catalysts) with low additives. Low protein binding coatings and treatments are not all the same, and while they may work at high protein concentrations and in the presence of common buffers, AntiBIND offers the superior protein recovery by as much as 3 fold at low protein concentrations and in the presence of harsh solvents.

Figure 3. The use of AntiBIND microplates has increased the percent recovery rate of sample by 50–300% over best-in-class low protein binding plates. The trend of increased sample recovery rate by using AntiBIND microplates over best-in-class microplates was observed with different proteins at different concentrations. (Data will be available in AntiBIND Tech. Note I.) When standard polypropylene plates are used at these low concentrations, the recovery rate after 24 hours was almost zero. The superior performance of AntiBIND microplates over best-in-class low protein binding plates and standard polypropylene plates across a range of proteins will allow for analytical reliability, which ultimately leads to cost savings, improved accuracy, and efficient allocation of valuable lab resources.

Peter Rezk (Peter.Rezk@wheaton.com) is global market manager at Wheaton.

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