Labeling antibodies with biotin or a fluorophore enables scientists to detect, track, and quantify that antibody and the molecules that it binds to. We cover many of the powerful experiments enabled by labeled antibodies in this article.

If you’re interested in performing one of these experiments, then you’ll need to know how to label antibodies. The good news is that labeling antibodies is pretty quick, easy, and efficient with Mix-n-Stain^TM antibody labeling kits.

Labeling antibodies with biotin or fluorophores involves three main steps: 1) preparing the antibody, 2) labeling the antibody, and 3) storing the antibody until you’re ready to use it. Proper execution of these steps is crucial for generating a reliable and versatile labeled antibody reagent. 

3 Steps of Labeling Antibodies 

1. Preparing the antibody  
2. Labeling the antibody 
3. Storing the antibody for future use

In this article we’ll briefly look at the Mix-n-Stain^TM antibody labeling protocol. We’ll also cover many of the common questions and issues that often come up when labeling antibodies.  

 

Table of Contents

Antibody labeling protocol overview

Ultrafiltration

Standard labeling protocol

Modified labeling protocol

What types of antibodies can I label?

Does my antibody have to be pure?

Choosing the right size of labeling kit

Tips and tricks

References

 

Antibody labeling protocol overview

In this section we’ll go over the antibody labeling protocol in detail and then discuss some frequently asked questions (FAQs) later in the article. If you’re looking for a step-by-step protocol on the Mix-n-Stain^TM labeling kit, you can find that here.

There are three main sections for the overall labeling protocol:

• Ultrafiltration (if necessary)
• Standard labeling protocol
• Modified labeling protocol

 

Ultrafiltration

Ultrafiltration prepares your antibody sample by concentrating your antibody into the desired range of 0.5 – 1.0 mg/mL and then depleting buffer components that interfere with the labeling reaction (Table 1).

This step is not always necessary. If your antibody is already at the recommended concentration of 0.5 – 1.0 mg/mL, and your buffer lacks any components that will hinder the labeling reaction (Table 1), then you can skip this step. In this case, your antibody is already prepared for the labeling reaction.

 

Table 1. Buffer Compatibility with Mix-n-Stain^TM Antibody Labeling Kits

Buffer Component	Compatibility
Sodium Azide	Compatible
Glycerol	Compatible with 10% glycerol or less Perform ultrafiltration step if greater than 10%
Tris	Compatible with 20 mM Tris or less Perform ultrafiltration step if greater than 20 mM
Glycine	Perform ultrafiltration
BSA or gelatin	Compatible with 4x greater than IgG or less Use modified protocol if greater than 4x
Ascites fluid	Use modified protocol
Serum	Not compatible, purify antibody first
Hybridoma cell culture supernatant	Not compatible, purify antibody first

 

However, if you need to concentrate your antibody, or if you need to deplete any buffer components that hinder antibody labeling, then a quick ultrafiltration step is the way to go.

The ultrafiltration vial includes a sample reservoir which is where you’ll pipet your antibody for concentration or buffer exchange. In this context, concentration means increasing the concentration of your antibody by reducing the buffer volume while keeping the number of antibodies the same. You can exchange the buffer during this step by increasing the volume again as described in a few paragraphs below.

There is a 10 kDa filter at the bottom of the sample reservoir. Molecules larger than 10 kDa, including your antibody, will be retained in the upper chamber while centrifuging the vial at 14,000 x g.

Smaller molecules, such as glycerol, tris, and glycine, will flow through the 10 kDa filter into the filtrate collection tube (Figure 1).

Figure 1. Antibodies and other large molecules (> 10 kDa) are concentrated in the sample reservoir (top), whereas small molecules flow the filter into the filtrate collection tube (bottom).

 

After each ultrafiltration step, when concentrating your antibody, you will remeasure its concentration to monitor whether it has reached the desired 0.5 – 1.0 mg/mL range. If you over-concentrate the protein, you can always dilute it back down with 1x PBS.

If you’re also doing a buffer exchange, then after you’ve concentrated your antibody down, add 1x PBS to the sample reservoir (Figure 2) and mix with the concentrated antibody before repeating centrifugation. This repeated concentration – adding buffer cycle will rapidly deplete buffer components that inhibit the labeling reaction to acceptable concentrations (Table 1).

Figure 2. 1x PBS solution being added to the sample reservoir with concentrated antibody to buffer exchange the antibody and deplete buffer components that hinder the labeling reaction.

Key tip: whenever removing your antibody solution from the sample reservoir chamber, use your pipette to gently rinse the membrane surface with the antibody-containing liquid. Proteins such as your antibody often stick to the membrane, so without this extra rinse step it may look like you’ve lost antibodies during the ultrafiltration step.  

Standard labeling protocol

Ok, so your antibody should now be at 0.5 – 1.0 mg/mL, and in a buffer without components that will impede the labeling reaction (Table 1). If your antibody is more concentrated than this, dilute it down to this range with 1x PBS.

Add the antibody solution to the 10x Mix-n-Stain^TM Reaction Buffer so that the final concentration is 1x. For example, you could combine 1 uL of 10x Mix-n-Stain^TM Reaction Buffer and 9 uL of your antibody solution. Gently pipette up and down a few times to mix the solution (Figure 3).

Transfer the entire Antibody-Reaction Buffer solution to the vial containing the fluorophore or biotin. Gently pipette up and down a few times, then cap the tube and gently invert a few times to make sure that all of the fluorophore or biotin is in solution.

Incubate the vial for 30 minutes at room temperature. If labeling the antibody with a fluorophore, perform this incubation in the dark and try to avoid light exposure as much as possible during subsequent steps (Figure 3).

Figure 3. Summarizes adding Mix-n-Stain^TM reaction buffer and then incubating with a fluorophore or with biotin. 9 uL of the antibody solution is added to 1 uL of 10x Mix-n-Stain^TM Reaction Buffer (1). The entire antibody-reaction buffer is added to a tube containing the fluorophore or biotin (2). The combined reaction is incubated at room temperature for 30 minutes (3).

 

 

Transfer the reaction into the storage buffer (Figure 4). The antibody is now ready to use for your experiments!

Figure 4. The standard labeling protocol includes ultrafiltration, labeling, and storage steps, in that order.

 

The labeled antibody is stable for at least 6 months when stored at 4 °C and protected from light. Alternatively, you can store the antibody in single-use aliquots at -20 or -80 °C for longer-term storage.

 

Modified labeling protocol

The modified labeling protocol is for antibodies that are in a solution with other proteins present. For example, BSA as a stabilizing agent or antibodies that were extracted in ascites fluid, both contain additional proteins besides the antibody. Most of the protocol is the same as the Standard Protocol, except for adding an ultrafiltration step before transferring the labeling reaction to the Storage Buffer.

So, after you’ve incubated the labeling reaction for 30 minutes, transfer it to the ultrafiltration vial. It’s ok if you previously used this vial for the same antibody during the first ultrafiltration step. Centrifuge the labeling solution at 14,000 x g, then use the desired volume of storage buffer to dilute, rinse the membranes, and remove the labeled antibody solution from the ultrafiltration vial (Figure 5). You can store the antibody solution as described above.

Figure 5. The modified labeling protocol includes an ultrafiltration step between the labeling and storage steps to keep the labeled antibody relatively concentrated since it will have less labeled molecules added per antibody.

 

What types of antibodies can I label?

Mix-n-Stain^TM Antibody Labeling Kits are optimized for IgG antibodies. While some researchers have used them to label other types of proteins (Trivedi et al, 2019), this use is not recommended by the manufacturer. In particular, the Mix-n-Stain^TM labeling conditions may cause denaturation of IgM antibodies.

If you do choose to label a protein other than an IgG antibody with the Mix-n-Stain^TM kit, it will be important to test the protein afterward to make sure that it is still functional or stable.

 

Does my antibody have to be pure?

While Mix-n-Stain^TM kits are optimized for labeling IgG antibodies, they will label most proteins. So, if your antibody is not pure, the labeling will be diluted between the antibody and the contaminating proteins. This will effectively limit the signal that you get out of your labeled antibody.

With pure IgG antibodies labeled as recommended, there should be approximately four to six fluorophore (or biotin) molecules added to each antibody. With contaminating proteins in the solution, you could get one, two, or even less than one label per antibody on average (Figure 6).

Figure 6. Pure antibodies (left) will have higher signal, biotin or fluorophores conjugated per antibody, compared to antibodies that are contaminated with other proteins (right).

 

If the signal from your labeled antibody is still high enough, then labeling contaminating proteins may not be an issue for your downstream assays. That’s because your antibody will bind to the antigen it’s detecting, whereas the contaminating labeled proteins can be washed away (Figure 7).

Figure 7. Antibodies in impure solutions can still be useful in experimental applications where the labeled contaminating proteins can be washed away.

 

Two common sources of impurities are BSA (bovine serum albumin) added as a stabilizing agent or antibodies that were extracted in ascites fluid which will contain additional proteins. You can still attempt to label antibodies in either of these conditions.

If the BSA added is less than four times greater than the concentration of the antibody, then just use the regular protocol that we discussed above. However, if the concentration of BSA is more than four times as much as the antibody, or if the antibody is in ascites fluid, then use the Modified Mix-n-Stain^TM labeling protocol.

If your antibody could use some more purification, another option is to purify it first using Protein A or Protein G affinity chromatography. This will separate your antibody from contaminating proteins which will improve the labeling efficiency on your antibody. When further purifying your antibody this way, make sure to limit components that inhibit the labeling reaction in your elution buffer (Table 1).  

 

Choosing the right size of labeling kit

Each type of labeling kit comes in three sizes:

-       5-20 ug

-       20-50 ug

-       50-100 ug

These values refer to the amount of antibody that you want to label. Keep in mind that you’ll want to use up the entire kit on one labeling reaction.

So, for instance, if you plan on labeling 10 ug of an antibody, you would want to use one of the 5-20 ug kits in its entirety. You would not want to use a fraction of one of the larger kits.

 

Tips and tricks for using GoldBio’s Mix-in-Stain^TM Antibody Labeling Kits

We’ve covered all of the basic steps and considerations already in this article, and below we go over some tips and tricks to help make your labeling reaction as successful as possible. Also, see this FAQ for even more information on how to successfully label your antibody.

·         There is no need to remove unconjugated free dye after the labeling step. While this is commonly done in other labeling systems, the manufacturer’s proprietary approach eliminates any need for it.

·         There is no concern for dye diffusion or transfer. The fluorophore, or biotin, is covalently linked to the antibody.

·         Mix-n-Stain^TM kits are optimized for use with IgG antibodies. Though there are reports of labeling other proteins  with these kits, we do not recommend this. If you choose to label other proteins with this kit, please perform a functional or biophysical check to make sure the protein was not denatured during labeling.

·         If the amount of antibody you want to label is right between two kit sizes, use the smaller kit to label your protein. For example, if you have 50 ug of antibody, use the 20-50 ug kit, not the 50-100 ug one.

·         Do not split the Mix-n-Stain^TM kits between multiple labeling reactions.

·         Antibody concentration and purity is very important. We recommend using a concentration between 0.5-1.0 mg/mL. Inaccurate protein concentrations or extensive impurities will hinder optimal reaction conditions.

·         If you’ve followed all of these steps but you don’t see any signal from your antibody, contact the antibody manufacturer and confirm that the formulation and concentration of your antibody is compatible with the labeling protocol for the kit that you’re using.

 

That’s a lot of detail to help make your antibody labeling as efficient as possible. If you’re ready to label your antibody, check out the plethora of options GoldBio has below as well as other resources to help you on your way.

 

 

References

Trivedi, P., Palomba, F., Niedzialkowska, E., Digman, M. A., Gratton, E., & Stukenberg, P. T. (2019). The inner centromere is a biomolecular condensate scaffolded by the chromosomal passenger complex. Nature cell biology, 21(9), 1127–1137. https://doi.org/10.1038/s41556-019-0376-4

 

 

 

Tags

antibody antibody labeling antibody purification Simon Currie

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