Introduction to Surface Functionalization of Biopolymers
Immobilizing (or covalently attaching) proteins, lipids, carbohydrates, and other polymers on biopolymer surfaces is incredibly important for a number of reasons. Want your surface to be hydrophobic or hydrophilic? Want to attach interesting fluorescent molecules on a sensor surface? There are a ton of possibilities! One of the most common uses for biopolymer surface functionalization is Surface Plasmon Resonance. Here, a protein is covalently attached to a gold surface and several different ligands are flowed past the protein-surface. Researchers can then study the binding and unbinding of ligands to proteins on the gold surface and determine on/off rates etc.
Another reason for immobilization of materials on a biopolymer surface might be to make it more hydrophilic. For a long time we have known that functionalizing long hydrophilic polymers on a surface can help prevent clotting and protein binding. This is one of the key methods for improving the blood compatibility of biomaterials. Without proteins to bind the surface and subsequent activation of platelets, biomaterials can be used inside the body for longer periods of time and they can even be implanted!
A Super-Simple EDC/NHS method for Surface Functionalization of Proteins on a Biopolymer
A common method for modifying the surface of a carboxyl-containing polymer with protein, is to attach the N-Terminus of the protein onto the surface. Here is a simple representation of the chemistry:
Materials for EDC/NHS Surface Immobilization of Proteins
Coupling Buffer: We need to make sure that your protein is neutrally charged using an appropriate buffer (and your knowledge of the isoelectric point, pI, of the protein). Make a buffer with 100 mM Formic acid (pH 3-4.5) , acetic acid (pH 4.0 – 5.5), or maleic acid (pH 5..5 – 7.0) in water. Use NaOH for pH equilibriation.
EDC [1-Ethyl-3-(3-dimethylamoniminopropyl) carboodiimide] at 0.4 M in water. Store at -20 C in small aliquots.
NHS [N-Hydroxysuccinimide] dissolved in water at 0.1 M. Store at -20 C in small aliquots.
Ethanolamine Hydrochloride dissolved in water at 1 M concentration, pH 8.5. Store at -20 C in small aliquots.
Your Protein of Interest at 50 ug/ml in an appropriate buffer.
HPLC, or high performance liquid chromatography is an amazing analytical technique for chemical compounds including biopolymers, small molecules, and polymers. In this method, a sample is first dissolved to make a solution. This solution is then injected into a “column” that contains resin that will interact with the sample. This will slow down the movement of the sample through the “column” and as the sample comes out the other side of the column, it is detected. This allows you to know both the time at which the sample comes out and the intensity of the sample that was detected. Here’s an overview of this technique:
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So, while there is continuous flow of some buffer through the column, we also inject our sample and observe as different molecules within the sample come out at different “retention times”. The detector on the end of the column can be any kind of detector but the most common types are refractive index (RI), ultraviolet (DAD), and fluorescence (FLD). Each of these will detect different properties of the molecules that come out of the column and display a chromatogram.
Types of Chromatography
Different column resin compositions determine the kind of chromatography that you are running and what molecules you can separate.
Normal Phase: The column is filled with silica particles which are polar and the buffer running through the system is non-polar. Once you inject your sample, polar particles will stick to the silica more and have a longer retention time than non-polar molecules.
Reverse Phase: The column is filled with hydrophobic particles (actually they are silica particles with long hydrocarbons on the surface). The buffer that is running through the system is polar (such as acetonitrile/water or methanol/water mixtures). This means that hydrophobic molecules will stick to the resin more and be retained longer.
Complete Step by Step HPLC guide
HPLC autosampler vials
I only use autosamplers since manual injection is tedious 🙂
Centrifugal filters with 0.2 um pores
To clean up samples
In a typical HPLC procedure you can decide the following variables:
What it does
With fast flow peaks come out sooner but there’s they’re harder to resolve and tend to blend together. For more resolution, run slower.
Affected by flow rate and solvent
Determines signal intensity, how quickly the peaks come out, signal fidelity
Determines the type of interaction with the sample
If using UV or FLD, you need to set the right excitation/emission wavelengths
Since HPLC is a very machine-variable technique, I can only provide general guidelines.
For sample preparation:
Dissolve your biopolymers or small molecules in a suitable solvent such as methanol
Centrifuge at 10,000 rcf in an eppendorf vial and keep the supernatant to remove any large particular matter
With a centrifugal filter, add 500 ul of your sample solution onto the top
Centrifuge at 10,000 rcf and collect the filtrate (the solution that successfully passes through the filter)
Load this sample into an HPLC vial
For setting up the HPLC machine:
Make sure you have all your buffers set up
Open the purge valve and purge the system for 5 minutes.
Add your samples into the autosampler tray
Stop the purge
Close the purge valve
Run the system at a normal flow rate (1 ml/min) with your buffer to equilibrate the column for 10 minutes
Make sure that your pressure is stable (ie, less than 2-3 bar of fluctuation)
Set up your sequence and your method
Run a standard before your actual samples or as part of the same sequence
Example buffer system to determine Fluoresceinamine levels in samples: Sample: Add 10 ug of fluoresceinamine into 1 ml of Acetonitrile. Buffer: Pure acetonitrile buffer on a C-18 column; this is “reverse phase”. Flow rate: 1 ml/min. Column: 4.6 mm x 30 cm size. Detection: Detect via a fluorescence detector set to Excitation @ 485 nm and Emission @ 535 nm.
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Notes on HPLC methodology
To clean the system and equilibriate it, you need to run enough solvent. However, this amount varies column-to- column. A typical 4.6 mm x 30 cm column should be clean when you follow the procedure above.
Isocratic means that the solvent concentration stays constant throughout the run.
It is useful to run standards before your samples as well as with your samples. Standards make it easy to identify which peak pertains to your molecule of interest.
Always use HPLC grade solvents. This is especially true for solvents like THF which are frequently sold with inhibitors that also complicate your ability to detect your molecule of interest.
Applications of HPLC on SciGine
HPLC is such a versatile technique. Take a look at these methods on SciGine which assay different types of chemicals in various samples.