Methods for Protein and Antibody Bioconjugation to Gold

We discuss methods for protein and antibody bioconjugation to gold including theory, alternative approaches, and protocols.

Gold isn’t just used to make pretty jewelry. It is a highly versatile reagent used in a wide variety of applications. One important application of gold is for protein and antibody bioconjugation. Gold has been conjugated to a range of proteins such as antibodies, protein A, lectins, enzymes, toxins, and many others. This article discusses methods for protein and antibody bioconjugation to gold. These include…

Methods for protein and antibody bioconjugation to gold include passive non-covalent coupling, ionic coupling, coupling via thiol-maleimide chemistry, streptavidin-biotin interactions, and via amide bond formation.

We’ve also discussed how to label proteins with fluorescent probes instead of gold in our articles.

Protein and antibody bioconjugation to gold nanoparticles - TEM image
Gold nanoparticles can be coated with polymers that include functional groups. These functional groups can be used for antibody and protein bioconjugation. TEM image of gold nanoparticles (source).

Applications of Protein- and Antibody- Gold Bioconjugates

Since the early 90s, gold-protein bioconjugates have seen significant research into their applications. The technology and understanding of these bioconjugates have developed considerably in the last 30 years, opening up applications in many fields, particularly in biomedical science.

Applications of gold bioconjugates include gold staining for electron microscopy, biological assays, and biosensors. 

Gold Staining for Electron Microscopy

Gold is an excellent material for electron microscopy because of its high electron density which allows it to produce high contrast images (to learn more about gold in microscopy check out this article from Fourie et al.). Once a protein is bioconjugated to gold, the gold acts as a stain, giving much better imaging of the target protein. Electron microscopy using gold bioconjugates has been studied most extensively for the immune system in the field of immunohistochemistry. Interestingly, by staining with gold particles of different sizes, multiple objects can be labeled in a single sample. 

Gold Bioconjugate Bioassays

Gold bioconjugates are now used in a wide range of bioassays because of their excellent ability to bind to specific proteins and their widely adjustable optical and surface properties. Versatility allows gold bioconjugates to be modified to fit specific experimental conditions with high precision. Depending on the configuration of the assay, the type of gold conjugate, and the target protein, potentially thousands of proteins can be assayed in parallel and assessed for their various interactions. Gasparyan et al. describe the use of gold bioconjugates in immunoagglutination and DNA hybridization in their review here.

Related articles:

  • Immunoprecipitation can also be utilized for some bioassays as an alternative to gold conjugate based bioassays. However, it’s a much more involved and tedious technique.

Gold Biosensors

Extending from their incredible versatility in bioassays, the use of gold bioconjugates as biosensors has been explored in extensive detail. The flexible optical properties of gold nanoparticles make them excellent optical biosensors once bound to a target protein. Other gold-based biosensors use gold’s electrochemical or piezoelectric properties to create biosensors for specific proteins. Historically, even radioactive gold particles have been used in biosensing, although the use of such bioconjugates has seen a significant decline for obvious reasons.

Gold biosensors provide several advantages compared to traditional techniques such as ELISAs – they can provide constant time-series information, used without taking time points, and assays are much cheaper after the initial biosensors have been developed. Gold nanoparticle biosensors are described in detail in this review article.

Immuno-gold labelling, TEM

Antibody bioconjugation to gold allows scientists to explore biological phenomenon using TEM using gold’s optical properties. Here’s an example of immune-gold labeling. TEM image of antibodies (orange) marked with gold particles (black dots) (source).

How Does Gold Bioconjugate to a Protein?

The bioconjugation of gold to a protein occurs through highly complex mechanisms which often vary depending on the type of gold, the functionalization of the gold, the target protein, and the local chemistry. While this seems overwhelming, these complex mechanisms can be simplified into two general types.

Types of protein bioconjugation to gold can be classified into two general types: passive conjugation which involves non-covalent interactions and covalent conjugation.

Type 1. Passive Bioconjugation of a Protein to Gold

Passive conjugation is the traditional method of conjugating gold to a protein. The interaction occurs passively between the protein and the gold particle through van der Waals and ionic forces. Depending on the conditions, a protein can spontaneously conjugate with a gold particle. By varying the size of the particle, and its ratio to the protein present, the conjugate can potentially bind multiple proteins to a single gold particle or surface.

Passive conjugation is useful because it’s a quick and easy way to produce a functional protein conjugate. However, it isn’t perfect. Passive conjugates often lack long-term stability and require very specific conditions for each protein used or conjugation may not occur. Worse, as conditions change, passively conjugated proteins can detach from the gold if the conditions in the experiment change.

Type 2. Covalent Conjugation of a Protein to Gold

It’s important to have a sensitive and stable conjugate that doesn’t decouple when the experimental conditions change. By covalently reacting your protein with gold particles, a significantly more stable gold bioconjugate can be formed. This allows for more complex experiments to be performed and potentially harsher conditions to be used. 

Covalent conjugation is achieved by using functionalized gold particles. These groups cross-link the protein to the gold creating a strong bond. However, covalent conjugation is usually a more involved process requiring a higher investment of time and money. Additionally, it requires pre-functionalized gold particles that you can react with.

For other protein conjugation chemistry methods, take a look at our article.

Methods for Antibody Bioconjugation to Gold Nanoparticles

Gold nanoparticles are an important type of metallic gold that is commonly used for bioconjugation with antibodies in immune system studies. Gold nanoparticles are metallic gold structures that are roughly 1 to 100 nm in size. Their outer layer can be customized to provide the desired functionality. 

Methods for antibody bioconjugation to gold nanoparticles include passive conjugation in water and EDC/NHS covalent conjugation.

Method 1. Passive Antibody Conjugation to Gold Nanoparticles:

As mentioned above, passive conjugation is a straightforward and easy technique to conjugate proteins to gold. This applies to antibodies and gold nanoparticles too. Here’s a step-by-step example of how to conjugate antibodies to gold nanoparticles based on the nanoComposix BioReady gold nanospheres:

Step 1. Gather your Starting Materials including Gold Nanoparticles, Antibodies, Clean Sample Tubes, and DI water.

Collect the appropriate gold nanoparticles, your target antibody, and sample tubes to perform the reaction in. You’ll need access to DI water. The nanoparticles should be suspended in DI water and the antibodies should be free of additional proteins or salt additives. 

Step 2. Mix the Antibodies with the Gold Nanoparticles in DI Water. 

Aliquot your gold nanoparticles into a sample tube. Rapidly add your purified antibodies to the gold nanoparticle solution and cover the sample tube.

Step 3. Incubate the Sample while Conjugation Occurs.

The conjugation reaction is quick but not instantaneous. Allow your sample to incubate at room temperature for 30 minutes or so with gentle stirring/rotating.

Step 4. Centrifuge the Sample and Collect the Conjugate. 

Your sample needs to be centrifuged at 3500 RCF for 10 minutes. After, carefully remove the supernatant. Resuspend your conjugate in DI water.

Step 5. Store your sample at 4°C / 39.2°F

Your sample needs to be stored at low temperatures to ensure it lasts as long as possible before decoupling. However, do not freeze your conjugate as this can cause the sample to decompose. 

Method 2. Covalent Conjugation of an Antibody to Gold Nanoparticles Using an NHS Ester Reaction.

Biomedical and nanomaterial suppliers often provide ready-made kits for performing protein conjugations to gold. These typically use EDC and NHS reactive groups to active carboxyl groups on activated gold nanoparticles which then couple to the protein. Stratech offers a kit that used carboxyl-activated gold nanoparticles to couple with antibodies using EDC/NHS chemistry. Here’s a step-by-step example of that process:

Step 1. Gather your materials: Carboxyl-activated gold nanoparticles, the target antibody, the conjugation reagent (EDC/NHS), buffer solutions, and suitable clean glassware.

Again, the antibody to be conjugated must be free of contaminants such as salts or other proteins. Make sure your glassware is clean and has been rinsed with DI water. 

Step 2. Prepare the conjugation reagent. 

Prepare the conjugation reagent in a buffer solution to ensure its stability. It should be prepared fresh, right before conjugation.

Step 3. Mix your gold nanoparticles in the conjugation reagent. 

This activates the gold nanoparticles and prepares them for conjugation. The mixture needs to be gently stirred while incubating at room temperature for 30 minutes.

Step 4. Add a coupling buffer to the solution and centrifuge the mixture for 30 minutes. 

The kit comes with a buffer for the coupling step. Carefully add that to the mixture and centrifuge it for 30 minutes. 

Step 5. Add your antibody to the mixture and sonicate for 10s. 

Carefully add the desired amount of antibody to the conjugation reagent and gold nanoparticles. Sonicate in a water bath sonicator for 10s. 

Step 6. Incubate the sample again for 2 to 4 hours at room temperature. 

Gently stir the mixture while it incubates so that no particles can settle at the bottom.

Step 7. Add more buffer solution and then centrifuge the mixture for 30 mins. Remove most of the supernatant. 

To ensure the stability of your conjugate, add more buffer solution, and then centrifuge the sample again for 30 minutes. Carefully remove most, but not all, of the supernatant. 

Step 8. Add a washing buffer and store conjugate at 4°C / 39.2°F ready for use. 

Finally, add a washing buffer to the solution and store it at 4°C / 39.2°F. Don’t freeze your conjugate.

Protein Bioconjugation to Gold Nanoparticles and Surfaces

Gold bioconjugates aren’t just used in immune system studies. Beyond antibodies, gold conjugates can be added to various proteins such as protein A, lectins, enzymes, toxins, and many others.

Methods for protein bioconjugation to gold nanoparticles include covalent conjugation using NHS/EDC reactions, covalent conjugation using thiol reactions, and chemisorption. 

Methods for protein bioconjugation to gold surfaces include physical conjugation to functionalized gold surfaces, dative binding of gold conducting electrons to amino acid sulfur atoms on the protein, and ionic interactions in polar solvents.

For more information on biopolymer surface functionalization techniques, take a look at our article.

Method for Protein Bioconjugation to Gold Nanoparticles via Thiol Reactions

Many proteins contain amino acids with sulfur atoms in their structure. These sulfur atoms can be reacted with functionalized gold nanoparticles to create thiol crosslinkers. Abcam produces a gold conjugate kit which uses gold nanoparticles to react with thiol-group containing proteins:

Step 1. Add the Protein into the Diluent Reagent.

Your protein must be free of additives such as other proteins or stabilizers.

Step 2. Add a Buffer to the Mixture.

Add the buffer slowly into the mixture with gentle stirring.

Step 3. Pipette the Sample Directly onto the Gold Nanoparticles.

The mixture should be resuspended gently by withdrawing and re-dispensing the liquid twice using a pipette. 

Step 4. Incubate the Sample for 60 Minutes.

Incubating the sample for longer has no negative effect on the conjugate.

Step 5. Add the Quenching Reagent to the Sample.

Slowly add the quenching reagent to the mixture with gentle stirring. The conjugate can be used after 20 minutes. 

Step 6. Store the Sample at 4°C / 39.2°F.

Don’t freeze the sample as it may damage the conjugate.

Method for Protein Bioconjugation to Gold Surfaces by Ionic Interactions

Proteins can be bioconjugated to gold surfaces using ionic interactions. This relies on the attraction between the negatively charged gold surface and the positively charged protein. The interactions can be adjusted based on the conditions in the mixture. This requires experimentation to optimize the conditions for conjugation to the surface. Once bound to the gold surface, these conjugates can potentially be used as biosensors or assays. Here’s a step-by-step method to help optimize your conjugate production based on an article by Rayavarapu et al.:

Step 1. Create Solutions Containing the target Protein

Ensure the protein is free from additives like salts or other proteins. 

Step 2. Add the Protein to the Gold Surface. 

Add to the gold surface with gentle mixing.

Step 3. Incubate the Sample for 60 Minutes

The sample will passively conjugate by ionic interactions. 

Step 4. Centrifuge the Sample and Remove the Supernatant.

The sample should be centrifuged at 3500 RCF for 10 minutes.

Step 5. Assess the Sample.

Analyze your sample to assess how well the protein has conjugated to your gold surface.

Step 6. Adjust the Conditions and Repeat the Experiment

Adjust the conditions of your reaction. This could be the concentration of protein, the pH of the solution, the incubation time, the temperature, etc. 

Streptavidin Gold Conjugation

Streptavidin is a protein purified from Streptomyces avidinii. It has a high affinity for biotin (vitamin B7), which is one of the strongest non-covalent interactions in nature. The strong interaction between streptavidin and biotin can be used to attach biomolecules, requiring harsh conditions to break the binding. By creating a gold-streptavidin bioconjugate, researchers gain access to a powerful tool for electron microscopy and the detection of biotinylated compounds. 

There are many methods to bioconjugate streptavidin to gold. 

To bioconjugate streptavidin to gold,  use EDC/NHS reactions to create covalent bonds or passively conjugate them in pH-sensitive conditions.  

Commercially Available Gold Conjugation Kits

Gold conjugation kits can be purchased from many nanomaterials and biomedical science suppliers such as Abcam, Nanocomposix, and Sigma Aldrich.

Kit NameKit SupplierHow does it work?Price in USD
Ab154873AbcamUses covalent conjugation and is designed to survive even the most extreme conditions. Gold particles are 40nm, 20 OD575
High sensitivity gold conjugation kitNanocomposixComplete kit containing everything needed to optimize a lateral flow assay.895
NHS ester functionalized conjugation kitCytodiagnostics – Sigma AldrichGold conjugate kit with NHS ester functionalized gold nanoparticles of 40 nm. Can be used to produce a SARS-CoV-2 conjugate for COVID-19 detection. 275

Protein Labeling with Fluorescent Probes – Theory and Methods

We discuss types of fluorescent probes, how to select a fluorescent probe for protein labeling, protein labeling kits, and protocols for protein labeling using kits.

Protein labeling is an extremely useful process in many fields including biology, biotechnology, medicine, forensics, genetics, and more. In simple terms, protein labeling is the use of a ‘label’ to bind to a protein in one way or another so that it can be detected, monitored, analyzed, and even purified. Being able to label a target protein opens scientists up to a range of possibilities for interacting with a target protein and understanding the many complex processes that proteins perform in the body. While there are many different types of protein label and labeling techniques, this article focuses on protein labeling with fluorescent probes.

Protein labeling with fluorescent probes can be accomplished by linking cyanine dyes, rhodamine dyes, or fluorescein to cysteines, lysines, tyrosines, or the N-terminus of your target protein.

What Are Fluorescent Probes?

Fluorescent probes are molecules that absorb light at a specific wavelength and emit it at a specific wavelength that can be detected. Fluorescent probes are also known as fluorescent tags or fluorescent labels.

Take a look at this page from Nature about fluorescent probes. The absorbance and fluorescence of a fluorescent probe are dependent on a range of factors including its chemical structure, the solvent it is dissolved in, its binding target, and more. Fluorescent probes are mainly used in biological studies, but can also be used in other applications. This includes tracers, dyes and stains, indicators, and more.

In protein labeling, fluorescent probes are typically a reactive derivative of a fluorescent molecule known as a fluorophore. Each specific fluorophore is chosen so that it will selectively bind to the target protein in the desired location. In some uses of fluorescent probes, the fluorophore isn’t always chemically bound to the protein and instead binds by other mechanisms. For example, some fluorophores are adsorbed into the protein binding sites and can be used to learn more about the binding site structure and its affinities. Here’s a great chapter from Methods in Cell Biology that explores protein labeling with fluorescent probes in more detail.

Protein labeling with fluorescent probes such as fluorescein can help analyze brain slices in mice
Green Fluorescent Protein (GFP) fluorescing in a mouse brain slice (source)

What Types of Fluorescent Probes Are There for Protein Labeling?

Since there is so much diversity in proteins, it of course makes sense that there is a huge range of fluorescent probes available to choose from. Organic fluorescent dyes are the most common way of labeling proteins. They are excellent choices for protein labeling because they can be fine-tuned by changing their structure to make them more target-specific or to adjust their fluorescence.

Types of fluorescent probes for protein labeling include cyanines, rhodamines, fluorescein, biological proteins like GFP, and quantum dots. 

Cyanine-Based Fluorescent Probes for Protein Labeling

These synthetic dyes contain conjugated polymethine chains with quaternary nitrogens as part of the system. Their fluorescent properties are easy to adjust depending on the functional groups and length of the conjugated chain. They often yield brighter and more stable fluorescence than alternative organic dyes. Here’s a great overview of cyanine dyes from Science Direct.

Related articles:

Rhodamine-Based Fluorophores

This family of dyes is mainly used for dying paper and as inks, but they also make excellent protein labels. They are high-performance dyes that are excellent for labeling antibodies in particular.

Fluorescein Labels for Proteins

The grandfather of organic fluorescent dyes for protein labeling. Fluorescein is one of the most important and successful fluorescent dyes. It is even listed as one of the WHO’s essential medicines.  There are countless fluorescein derivatives in a huge range of applications so they remain the kings of protein labeling.

Biological Fluorophores

These fluorophores are formed from biological structures that can fluoresce. While they are often more expensive and time-consuming to use, they can be bound to proteins very effectively in specific cases and introduced into living cells, bacteria, or even entire organisms. Biological fluorophores have the advantage of being less likely to result in issues of toxicity by negatively affecting the proteins they are bound to. They can be made from other proteins, enzymes, antibodies, or other common biological structures that can be bound to a protein.

Quantum Dots 

These relatively new fluorophores are significantly brighter and more stable than organic fluorescent dyes. They are tiny (nanometer scale) crystals made from semiconductors. Their fluorescence is linked to their size and shape, and they are exceptionally stable (one study reported quantum dots fluorescing for 4 months in vivo!). However, they are still novel technology and need more research. The main challenge with quantum dots is their toxicity. Since they are made from heavy metals and have high stability, they are potentially very toxic depending on a range of parameters such as their size, shape, composition, and more. They are powerful tools for protein labeling as they can be coated in different ways to optimize their binding. 

Quantum dots are stable fluorescent probes for protein labeling
The fluorescence wavelength of quantum dots can vary depending on their size (source).

Tips to Select A Fluorescent Probe for Protein Labeling

Proteins are large, complicated biological structures with potentially thousands of relevant functional groups and binding sites, as well as a specific 3D structure. This means that choosing the right fluorescent probe for your target protein is essential. However, there isn’t always literature on every protein and fluorophore, so you’ll have to do some experimentation to make sure you get the right fluorescent probe for the job.

To select a fluorescent probe for protein labeling, choose a fluorescent probe that’s specific to your protein, in a detectable wavelength, stable in your experimental conditions, and doesn’t interfere with other fluorophores or components in your experiment.

1. Determine What Protein You Want to Label

This seems obvious but it’s the most important factor. You need to know exactly what you’re looking for, or there isn’t much point trying to label a protein. Are you targeting a specific family or protein? Or one particular protein? The more you know about your target, the easier it will be to choose a fluorescent probe. You’ll also need to analyze how to purify your recombinant protein with your fluorescent label.

2. Determine if Experimental Conditions Will Quench or Interfere With a Fluorophore

What are you trying to do once you’ve labeled your target protein? Monitoring intracellular protein processes in real-time might require a very stable fluorescent probe that has low toxicity. What if you’re trying to purify your target protein? That might open your options to include fluorescent probes that aren’t as long-lived but are brighter and will give you more precision.

3. Choose a Fluorophore That Binds Specifically to Your Target Protein

Your fluorophore needs to be specific in binding to your target protein. It’s no use to you if your label binds to various proteins that you aren’t interested in and gives you false readings. Worse yet, a poorly chosen fluorophore might bind to multiple proteins at once. We’ve discussed protein conjugation chemistry in detail in another article.

4. Find a Fluorophore That Can Be Detected Easily 

Great, you’ve added your fluorescent probe to your protein, but can you see it? Your fluorescent probe needs to be bright and easy to detect in your sample. If your sample matrix affects the fluorescent of your probe, you’re not going to get an accurate measurement. It’s especially important that it doesn’t fluoresce at the same wavelength as any component of your sample of you’re going to get a false reading.

5. Ensure That Your Fluorophore Is Stable in Your Experimental Conditions

Your fluorescent label of choice needs to be stable in your sample and when bound to your target protein. It will need to remain stable long enough for you to complete your experiment, but without it affecting the biological system you’re monitoring.

6. Minimize Interference Between Your Fluorophore and Other Fluorophores or Experiment Components

In many cases, your fluorescent probe needs to not interfere with the function of the protein, or the function of the biological system you’re monitoring the protein in. There’s no point labeling a protein if its function is completely impaired by the binding of your label. Worse yet, you won’t be able to monitor proteins in cells or other living organisms if your probe is so toxic that it kills them. When working with living samples, make sure to select a probe with suitable toxicity for the duration of your experiment.

Techniques to Analyze Proteins Labeled With Fluorescent Probes

Once you’ve labeled your protein with a fluorescent probe, there’s an amazing range of potential uses for your labeled protein. Proteins can be observed, quantified, and studied in great detail once you’ve attached a label.

To analyze your protein labeled with a fluorescent probe, you can use techniques such as fluorescent microscopy to quantify and monitor your target protein, flow cytometry to sort proteins, and even monitor living cells using fluorescent live-cell imaging with multiple labeled proteins. 

Analyzing Proteins Labeled With Fluorescent Probes Using Fluorescence Microscopy 

Labeled proteins can be identified in cells and cellular components with exceptional specificity. Many uses extend from this. For example, the levels of proteins expressed in certain tissue can be quantified to understand the effects of a specific gene. Another example is the use of multiple fluorescent probes to monitor a protein and the components it interacts with to provide more detail about the mechanisms occurring within a cell. In medical diagnostics, cancer-specific proteins can be labeled to learn more about specific cancers. We’ve written about immunofluorescence microscopy in detail and discussed how to use antibodies attached to fluorescent probes.

Flow Cytometry Can Sort Proteins Labeled With Fluorophores

Labeled proteins can be sorted and quantified in real-time as they pass through a light beam of detectors that measure their fluorescence. In medicine, this technique can be used to rapidly screen for medically relevant proteins. This technique is used in immunology (for example, antibodies), hematology (blood proteins and other markers), oncology (cancer-specific proteins as mentioned above), and even genetics (measuring gene expression by protein markers). Flow cytometry is a popular technique because it is relatively cheap and reliable. We’ve discussed the flow cytometry method and theory behind this technique here.

Live-Cell Imaging Can Be Used to Visualize Proteins Labeled With Fluorescent Probes in Biological Systems

Labeled proteins are extremely useful in monitoring intracellular processes in real-time using time-lapse fluorescence microscopy. It provides insights into the life of a cell and the active processes within. As mentioned above, the use of multiple fluorescence probes can provide intricate details to the internal structure of a cell. However, a careful balance needs to be found between collecting enough data without producing phototoxic effects from overusing the fluorescent probes.

Related articles:

  • Fluorescent western blotting is a simple and effective technique for analyzing labeled proteins

Where Can I Buy Fluorescent Probes?

Most major chemical suppliers sell specific fluorophores. Organic fluorescent dyes are the most commonly available types but now more unusual types like quantum dots are becoming available.  Many suppliers produce protein labeling kits that contain everything needed to complete the process in a short time. Some offer the ability to label up to 10 mg of protein.

Fluorescent probes can be bought from most life science suppliers such as Anaspec, ThermoFisher, and LiCor.

Here are some examples of protein labeling kits found on the market:

AnaTag 5 microscale protein labeling kits. These labeling kits use fluorescein isothiocyanate (FITC) as a fluorophore. The kit is suitable for biological applications and can label 3 x 200 ug of protein.

Alexa Fluor protein labeling kits from ThermoFisher. These kits offer a very straightforward way to covalently label 1 – 10 mg of protein with a fluorescent dye. They offer a range of dyes with a broad range of excitation and emission wavelengths.

LI-COR IR Dye protein labeling kits.  These kits are designed for labeling antibodies for use in flow cytometry where fluorophore-conjugated antibodies are required.

Step-by-step Example of Labeling a Protein With a Fluorescent Probe

The most straightforward way to label a protein with a fluorescent probe is to use a protein labeling kit like those mentioned above. Most of these processes use a similar process to label the proteins. Below you’ll find the general steps that protein labeling kits like this one. They are very easy to use and require a small time investment. 

Materials Needed for Protein Labeling

·        Protein labeling kit

·        A suitable amount of purified protein

·        30 minutes of hands-on time

·        2 hours until the protein is ready

General Steps to Label With Fluorescent Probes

Step 1. Add Your Protein Into a Vial With the Fluorophore and a Magnetic Stir Bar 

Most kits come with a premeasured quantity of dye and vial for you to perform this step.

Step 2. Let the Reaction Occur With Gentle Stirring 

The kit will tell you how long you need to stir the reaction. Be careful not to stir too aggressively as some proteins are sensitive to agitation.

Step 3. Purify the Protein Using the Size Exclusion Column Provided 

These are usually gravity-fed size exclusion columns that purify out your protein.

Step 4. Collect Your Purified Labeled Protein

Collect your protein from the column. Now you can perform your experiments on the protein.

As you can see, the process is fairly simple and requires minimal effort as all of the equipment you need to label the protein is provided in the kit.

Protein Conjugation Chemistry – Theory and Examples

We discuss protein conjugation chemistry techniques such as lysine, cystein, or site-specific conjugation using bifunctional linkers.

Before we do a deep dive into protein conjugation chemistry, let’s first describe what a conjugated protein is.

A conjugated protein is a protein to which another chemical group or molecule has been attached. Typically, covalent bonding interactions are used to conjugate molecules to proteins.

Proteins that contain only amino acids are generally referred to as free proteins

Conjugated proteins can be attached to carbohydrates, lipids, organic complexes and may even have stabilized metal ions. 

Conjugated proteins contain free proteins, as defined earlier, attached to molecules, called prosthetic groups.  When carbohydrates are bound to a protein, the protein is called a mucoprotein or glycoproteins (considered to be the most abundant and largest conjugated protein). Some categories of conjugated proteins also take the form of proteolipids or lipoprotein. Proteolipids have the tendency to behave typically like lipids and are quite soluble in organic solvents. Lipoproteins (lipids attached to a protein molecule) behave typically like a protein. The lipid inside a lipoprotein imparts a lower density to the protein; which means a lipoprotein has a relatively low physical weight.

You can also have Metalloproteins and Chromoproteins, which are other derivatives of conjugated proteins. A metalloprotein has a metal group specifically bound to an amino acid moiety. A chromoprotein is a colored protein composed of a protein and a chromophore (functionality that gives them color).

A typical metalloprotein called hemoprotein is represented below. Note the way the iron ion is bound.

A metalloprotein that has conjugated iron

 

Hemoglobin is a conjugated metalloprotein that binds iron. Image from ScienceDirect.

Related articles:

Bifunctional Reagents for Protein Conjugation Chemistry

A bifunctional reagent features reactive or some very active end groups or molecules that make bioconjugation (crosslinking) possible. These bifunctional reagents can actively bind to compatible functional groups through their active end groups. 

Research has shown four main functional groups make effective target moieties on proteins and prosthetic molecules. These functional groups include;

  • Primary amines: A primary amine has an amino functional group (- NH2). We can have the aliphatic amine (R-NH2) and the Aromatic amine (Ar-NH2). Aromatic amines are more reactive and easier to conjugate to proteins than aliphatic amines. 
  • Carbonyls: Identified by the carbonyl functional group (R-C=O).
  • Thiols (sulfhydryls): This is a functional group that has sulfur bonded to a hydrogen atom. They are usually denoted as R-SH.
  • Carboxyls: Denoted R–COOH. 

A closer look at all four bifunctional groups shows that they are also a part of amino acids, so they are easy to find on proteins. 

protein conjugation chemistry using cystine

Some common homo and hetero bifunctional reagents for protein conjugation. Image from SpringerLink.

Homobifunctional Reagents for Protein Chemistry vs. Heterobifunctional Crosslinkers

Bifunctional reagents can be homobifunctional or heterobifunctional. 

Homobifunctional reagents have the same reactive group on either side and can be utilized for one-step protein conjugation chemistry. Heterobifunctional reagents have different reactive groups on each side and are typically utilized in multistep conjugation reactions with proteins. 

Researchers have also utilized homobifunctional crosslinkers with N-hydroxylsuccinimide (NHS) esters to identify previously unknown interactions between proteins. 

How to Use Bifunctional Reagents For Protein Conjugation

If you’re wondering how to utilize a bifunctional reagent for protein conjugation, in general you need to:

To use a bifunctional reagent for protein conjugation, first determine the target moiety on your protein and your prosthetic molecule. Then find a bifunctional reagent that will bind both your free protein and your prosthetic molecule. Finally, utilize the appropriate protein conjugation conditions to attach the protein to your linker and then conjugate your protein, with the crosslinker, to the prosthetic molecule.

Here are more details on the above general steps. 

Step 1. Determine A Target Moiety On Your Protein First

Amino acids on proteins consist of carboxyl groups and amino groups. Since proteins are linear polymers consisting of amino acids, one end of the protein will have an unconjugated carboxyl and the other end will have an unconjugated amine group. The carboxyl end is called the C-terminus and the amine end is called the N-terminus. Both of these ends can be conjugated to. Protein labeling with fluorescent probes is a common reason to utilize the N- and C- termini.

You can quantify the N-terminus along with any free amine groups in a protein by reacting the protein with fluorodinitrobenzene (FDNB) or dansyl chloride. The fluorodinitrobenzene (FDNB) or dansyl chloride will link with any free amine inside the protein. If you notice that there are a lot of amines, then consider using bifunctional linkers with at least one NHS group to react to the amines on the protein. See below for lysine conjugation chemistry.

Other viable target moieties on your protein could include thiols from cysteine residues or the C-terminus carboxyl group. See the image below for amino acids that are easy to react with. 

lysine, cysteine, aspartic acid, and glutamic acid are excellent for making protein conjugates

Lysine, Aspartic Acid, Cysteine, and Glutamic Acid are good target moieties for protein conjugation reactions due to their amine and carboxyl side chains. Image from NEB.    

Step 2. Determine a Target Chemical Moiety on Your Prosthetic Molecule

Conjugated proteins are proteins linked with chemical groups on other molecules, called prosthetic molecules. Prosthetic molecules can be carbohydrates, lipids or even ions. 

You need to determine which chemical moiety on your prosthetic molecule you will conjugate to the protein of interest using the bifunctional reagent. 

The best moieties on your prosthetic molecule to bind to are amines, carboxyl groups, or sulfhydryls as we stated previously. 

If you can install an azide, tetrazine, or alkyne on your prosthetic molecule, these groups can make your protein conjugation chemistry really easy. 

If none of these groups are available on your prosthetic molecule, consider converting a hydroxyl to an aldehyde to make it more reactive.

Typically nanoparticles that are commercially purchased can include functionalized polymer surfaces that you can conjugate to proteins. We discuss protein and antibody bioconjugation to gold in our article.

click chemistry is commonly utilized for conjugating proteins

Azide-alkyne ‘click’ chemistry is a common method utilized for protein conjugation chemistry. It’s considered ‘bioorthogonal’ because azides and alkynes are uncommon in proteins. Image from Royal Society of Chemistry.

Step 3. Find a bifunctional reagent that can react with both chemical moieties

Bifunctional reagents are important in the protein conjugation process as we’ve discussed previously. 

The bifunctional reagent that you use to conjugate your protein to the prosthetic molecule must include chemical groups that can react with your protein (from step 1) and chemical groups that can react with your prosthetic molecule (from step 2).

If the two groups from Step 1 and Step 2 above are different, you need to use a heterobifunctional reagent. Western blotting can be utilized to prove that your conjugation reactions yielded higher molecular weight results.

Using heterobifunctional reagents for protein conjugation chemistry

To use a heterobifunctional reagent, first react it with the protein. This reduces steric hindrance compared to the opposite case where you first react with the prosthetic group because your linker bifunctional reagent will be able to reach deeper parts of your protein without the prosthetic group interacting with the protein. 

After reacting with the protein, you can react to the other functional group on the bifunctional reagent to react with your prosthetic molecule. 

Note that two steps are involved for heterobifunctional reagents in order to avoid polymerization or side linkage of functional groups. 

Using homobifunctional reagents for protein conjugation

Homobifunctional reagents can be utilized in one pot reactions containing both the protein and the prosthetic group or separately in two different conjugation reactions. 

Site-specific protein conjugation

For site specific protein conjugation, researchers typically include unnatural amino acids inside their protein during expression. Cysteine groups could be used for site specific conjugation if only one disulfide bond is present in the protein and hence only those sulfhydryl groups are accessible to your bifunctional reagent.

Bioorthogonal reactions for protein conjugation

Azids, ketones, and aldehyde can be coupled to a protein via bioorthogonal chemistry. Typically, bifunctional reagents utilized to conjugate to these won’t react with amines or carboxyls on the protein. Ketones and aldehydes can be reacted with aminooxy or hydrazide compounds to yield a very stable oxime or hydrazone linkage between the protein and the biomolecule. 

Lysine Conjugation Chemistry

Lysines are the most common targets for protein conjugation chemistry reactions. The amine on the lysine is essentially a nucleophile and reacts to amine-reactive chemicals such as N-hydroxysuccinimide (NHS) ester (these react via lysine acylation).

For successful Amine-NHS conjugation reactions, the pH of the reaction solution must be below 10.5, which is the pka of the ammonium group in lysine. This helps deprotonate the lysine groups. This means that the reaction should be carried out at ph around 8.5 to 9.0. 

Lysine may also react with isothiocyanates and isocyanates and benzoyl fluorides.

Advantages of lysine-based conjugation

  • Simple
  • Commonly utilized for antibody conjugation
  • Highly reactive, if they are accessible
  • Versatile because it can react several cytotoxic agents (especially onto antibodies)

Bioconjugation strategies for lysine residues on proteins. Image from Nature

Maleimide Protein Conjugation Reaction Chemistry

You can react cysteines in proteins with maleimide groups as long as your buffer is in a pH between 6.0 and 7.0. A common approach is to reduce disulfide bonds in proteins first with DTT or TCEP and then to react with a maleimide-containing linker.

At a pH between 6.0 to 7.0, the maleimide group can react with sulfhydryl groups resulting in the production of a stable thioether linkage and the reaction is irreversible. At pH above 8.0 (alkaline), say 8.5, the reaction seems to favor primary amine and the rate of hydrolysis of the maleimide group to maleimide acid (not reactive) will increase.  

You must eliminate compounds that contain thiol groups, like dithiothreitol (DTT) and β-mercaptoethanol (BME, aka 2-mercaptoethanol), from the reaction because they tend to compete with thiol groups on proteins. For instance, if you utilized dithiothreitol (DTT) to reduce disulfides in a protein and open up the sulfhydryl groups, you would need to thoroughly remove the DTT using a desalting column before you start the maleimide reaction. Instead of DTT, consider using the disulfide-reducing agent TCEP which lacks thiols. 

You can quench free maleimide left over after reacting your protein by adding free thiols. You can also check your conjugation reactions using our HPLC Step By Step guide.

Advantages of Maleimide Protein Conjugation

  • Thiols are only present in proteins on Cysteines
  • For many proteins, one or two disulfide bonds exist, which mean that you can reduce them and conjugate in a site-selective manner using thiol-maleimide chemistry
  • Cysteines can be readily introduced into proteins without affecting function, using site directed mutagenesis

Here are some strategies for using thiol-maleimide protein conjugation chemistry to create cleavable and permanent antibody drug conjugates. Image from RSC.

Protein conjugation protocol example

There are several protein conjugation kits that are available. Here’s an example from Vector labs. However, you can also utilize the chemical reactions above to create protein conjugates. 

The steps below show a typical example of conjugating to lysines on a protein, based on information from GBiosciences

Materials

  • A PEG linker containing NHS such as any of these
  • DMSO or DMF 
  • Amine free buffer at pH 7-8. Phosphate buffer works well
  • 0.5-1.0 NaOH
  • Protein containing Amines
  • Desalting column

Procedure

  1. Dissolve 1-2mg the NHS ester reagent in 0.25 ml of DMSO or DMF
  2. Then add 2ml of phosphate buffer to the same reaction vial
  3. Add 100µl 0.5-1.0N NaOH to 1ml of the reaction from step 2.  Vortex for 30 seconds
  4. Check that the pH is between 8.5 and 9
  5. Add your protein in pH 8.5-9 phosphate buffer to this vial
  6. Use a desalting column to remove any unreacted linker molecules. Here’s a step by step method for protein purification of recombinant proteins.
  7. Utilize the other end of the PEG linker to conjugate a prosthetic molecule