Name: antibody labeling with fluorescent dyes using magnetic protein a and protein g beads
Uploaded: 2016-09-15T14:00:00
Description: Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

1. Antibody Capture onto High Capacity Magnetic Protein A or Magnetic Protein G Beads
<ol>
	<li>Uniformly re-suspend magnetic beads by gentle shaking. Keep the suspension uniform when aliquoting beads.</li>
	<li>Add 50 &#181;l of bead slurry to a 1.5 ml microcentrifuge tube. Place in the magnetic stand for 10 sec. Carefully remove the storage buffer.</li>
	<li>Add 250 &#181;l of antibody binding buffer.</li>
	<li>Mix and place in the magnetic stand for 10 sec. Carefully remove the binding buffer.</li>
	<li>Add 1.0 ml o...

<ol>
	<li>Colwill, K., Graslund, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+roadmap+to+generate+renewable+protein+binders+to+the+human+proteome.">A roadmap to generate renewable protein binders to the human proteome.</a> Nat Methods. 8 (7), 551-558 (2011).</li><li>Silverstein, A. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Labeled+antigens+and+antibodies:+the+evolution+of+magic+markers+and+magic+bullets.">Labeled antigens and antibodies: the evolution of magic markers and magic bullets.</a> Nat Immunol. 5 (12), 1211-1217 (2004).</li><li>Day, J. J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemically+modified+antibodies+as+diagnostic+imaging+agents.">Chemically modified antibodies as diagnostic imaging agents.</a> Curr Opin Chem Biol. 14 (6), 803-809 (2010).</li><li>Cunningham, R. E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overview+of+flow+cytometry+and+fluorescent+probes+for+flow+cytometry.">Overview of flow cytometry and fluorescent probes for flow cytometry.</a> Methods Mol Biol. 588, 319-326 (2010).</li><li>Eaton, S. L., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+guide+to+modern+quantitative+fluorescent+western+blotting+with+troubleshooting+strategies.">A guide to modern quantitative fluorescent western blotting with troubleshooting strategies.</a> J Vis Exp. (93), e52099 (2014).</li><li>Dundas, C. M., Demonte, D., Park, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Streptavidin-biotin+technology:+improvements+and+innovations+in+chemical+and+biological+applications.">Streptavidin-biotin technology: improvements and innovations in chemical and biological applications.</a> Appl Microbiol Biotechnol. 97 (21), 9343-9353 (2013).</li><li>Kraeber-Bodere, F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Radioimmunoconjugates+for+the+treatment+of+cancer.">Radioimmunoconjugates for the treatment of cancer.</a> Semin Oncol. 41 (5), 613-622 (2014).</li><li>Leal, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-drug+conjugates:+an+emerging+modality+for+the+treatment+of+cancer.">Antibody-drug conjugates: an emerging modality for the treatment of cancer.</a> Ann N Y Acad Sci. 1321, 41-54 (2014).</li><li>Drachman, J. G., Senter, P. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-drug+conjugates:+the+chemistry+behind+empowering+antibodies+to+fight+cancer.">Antibody-drug conjugates: the chemistry behind empowering antibodies to fight cancer.</a> Hematology Am Soc Hematol Educ Program. 2013, 306-310 (2013).</li><li>Hermanson, G. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Bioconjugate Techniques. , 380-382 (1996).</li><li>Shrestha, D., Bagosi, A., Szollosi, J., Jenei, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparative+study+of+the+three+different+fluorophore+antibody+conjugation+strategies.">Comparative study of the three different fluorophore antibody conjugation strategies.</a> Anal Bioanal Chem. 404 (5), 1449-1463 (2012).</li><li>Vira, S., Mekhedov, E., Humphrey, G., Blank, P. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fluorescent-labeled+antibodies:+Balancing+functionality+and+degree+of+labeling.">Fluorescent-labeled antibodies: Balancing functionality and degree of labeling.</a> Anal Biochem. 402 (2), 146-150 (2010).</li><li>Isa, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+screening+system+to+identify+small+molecules+that+induce+internalization+and+degradation+of+HER2.">High-throughput screening system to identify small molecules that induce internalization and degradation of HER2.</a> ACS Chem Biol. 9 (10), 2237-2241 (2014).</li><li>Liao-Chan, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+assessment+of+antibody+internalization+with+novel+monoclonal+antibodies+against+Alexa+fluorophores.">Quantitative assessment of antibody internalization with novel monoclonal antibodies against Alexa fluorophores.</a> PLoS One. 10 (4), e0124708 (2015).</li><li>Riedl, T., van Boxtel, E., Bosch, M., Parren, P. W., Gerritsen, A. F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-Throughput+Screening+for+Internalizing+Antibodies+by+Homogeneous+Fluorescence+Imaging+of+a+pH-Activated+Probe.">High-Throughput Screening for Internalizing Antibodies by Homogeneous Fluorescence Imaging of a pH-Activated Probe.</a> J Biomol Screen. 21 (1), 12-23 (2016).</li><li>Nath, N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Homogeneous+plate+based+antibody+internalization+assay+using+pH+sensor+fluorescent+dye.">Homogeneous plate based antibody internalization assay using pH sensor fluorescent dye.</a> J Immunol Methods. 431, 11-21 (2016).</li><li>Lundberg, E., Sundberg, M., Graslund, T., Uhlen, M., Svahn, H. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+method+for+reproducible+fluorescent+labeling+of+small+amounts+of+antibodies+on+solid+phase.">A novel method for reproducible fluorescent labeling of small amounts of antibodies on solid phase.</a> J Immunol Methods. 322 (1-2), 40-49 (2007).</li><li>Strachan, E., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Solid-phase+biotinylation+of+antibodies.">Solid-phase biotinylation of antibodies.</a> J Mol Recognit. 17 (3), 268-276 (2004).</li><li>Dezfouli, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+bead+assisted+labeling+of+antibodies+at+nanogram+scale.">Magnetic bead assisted labeling of antibodies at nanogram scale.</a> Proteomics. 14 (1), 14-18 (2014).</li><li>Nath, N., Godat, B., Benink, H., Urh, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=On-bead+antibody-small+molecule+conjugation+using+high-capacity+magnetic+beads.">On-bead antibody-small molecule conjugation using high-capacity magnetic beads.</a> J Immunol Methods. 426, 95-103 (2015).</li><li>Lund, L. N., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Exploring+variation+in+binding+of+Protein+A+and+Protein+G+to+immunoglobulin+type+G+by+isothermal+titration+calorimetry.">Exploring variation in binding of Protein A and Protein G to immunoglobulin type G by isothermal titration calorimetry.</a> J Mol Recognit. 24 (6), 945-952 (2011).</li><li>Safarik, I., Safarikova, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Magnetic+techniques+for+the+isolation+and+purification+of+proteins+and+peptides.">Magnetic techniques for the isolation and purification of proteins and peptides.</a> Biomagn Res Technol. 2 (1), 7 (2004).</li><li>Flygare, J. A., Pillow, T. H., Aristoff, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody-drug+conjugates+for+the+treatment+of+cancer.">Antibody-drug conjugates for the treatment of cancer.</a> Chem Biol Drug Des. 81 (1), 113-121 (2013).</li><li>Shen, B. Q., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Conjugation+site+modulates+the+in+vivo+stability+and+therapeutic+activity+of+antibody-drug+conjugates.">Conjugation site modulates the in vivo stability and therapeutic activity of antibody-drug conjugates.</a> Nat Biotechnol. 30 (2), 184-189 (2012).</li><li>Acchione, M., Kwon, H., Jochheim, C. M., Atkins, W. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Impact+of+linker+and+conjugation+chemistry+on+antigen+binding,+Fc+receptor+binding+and+thermal+stability+of+model+antibody-drug+conjugates.">Impact of linker and conjugation chemistry on antigen binding, Fc receptor binding and thermal stability of model antibody-drug conjugates.</a> MAbs. 4 (3), 362-372 (2012).</li></ol>

The goal of this study was to develop a method to label antibodies, present in the cell media at low concentrations, with a variety of small molecules. Such a method will allow a large number of antibodies, during the early stages of antibody discovery, to be labeled with small molecules and screened using a biologically relevant assay. One such assay is the receptor-mediated antibody internalization assay where internalization may vary between antibodies even with similar binding affinities. Hence, it is important to sc...

Antibodies labeled with small molecules are perhaps the most commonly used reagents in biology1,2. Antibodies labeled with fluorescent dyes and biotin are extensively used in imaging, immunoassays, flow cytometry, western blots, and immunoprecipitation among other applications3-6. Radiolabeled antibodies3,7 find extensive use in imaging and therapy, antibodies labeled with cytotoxic drugs (ADCs) are offering new options for the treatment of cancers, and two ADCs have already been approved for therapeutic use8. In spite of their extensive use, the methods for labeling antibodies have remained surprisingly unchanged and typically involve multiple reactions and desalting steps9-12. Solution methods work very well in cases where only a few antibodies need to be labeled and are available in highly purified form at high concentration and in sufficient volumes. However, for newer applications like ADCs, there is a need to label antibodies at the early hybridoma stage so that they can be screened for biologically relevant properties, for example, receptor-mediated antibody internalization13-16. At the hybridoma stage, sample volumes are limited, antibodies are expressed at low concentrations and a number of samples are large, hence solution based labeling methods are not suitable.
To simplify and improve the throughput of the traditional antibody labeling methods, a few alternative approaches have been proposed17,18. One approach is to use non-magnetic Protein A affinity beads packed in small columns to capture antibodies followed by the labeling reaction and elution of labeled and purified protein. This method can be used to label antibodies directly from cell media, however, the use of columns can be laborious. A magnetic bead based method has recently been reported19 that eliminates the use of columns and improves throughput but due to the limited antibody binding capacity of the beads, only nanogram to low microgram quantities of the antibodies could be labeled.
We recently developed and used high capacity magnetic Protein A and Protein G beads (&#62;20 mg of Human IgG/ml of settled beads) to label antibodies present in cell media with small molecules20. The high capacity of the beads allows tens to hundreds of micrograms of antibody to be labeled conveniently and the rapid magnetic response of the beads simplifies handling and processing of a large number of samples in parallel. Using fluorescent dyes as surrogates for small molecules, we show that the method is compatible with amine and thiol labeling chemistry and offers high recoveries of labeled and very pure antibodies.
This protocol and the accompanying video describe on-bead labeling of mouse antibodies present in the cell media using Magnetic Protein A and Protein G beads. The protocol is divided into four sections: Section 1 describes the capture of antibodies onto the bead from biological samples. Following capture, the labeling of antibodies with fluorescent dye using amine chemistry or using thiol chemistry is described in sections 2 and section 3, respectively. Finally, section 4 describes the method for the calculations of the antibody concentration and the dye to antibody ratio.

<table border="0" cellpadding="0" cellspacing="0" width="592">
	<tbody>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein A Beads</td>
			<td style="width:103px;">Promega Corporation</td>
			<td style="width:119px;">&#160;G8781</td>
			<td style="width:155px;"></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Magne Protein G Beads</td>
			<td style="width:103px;">Promega Corporation&#160;</td>
			<td style="width:119px;">&#160;G7471</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-SE (Succinimidyl Ester)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20001</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 532-ME (Malemide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A10255</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">AlexaFluor 647-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">A20347</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:216px;">Fluorescein-ME (Maleimide)</td>
			<td style="width:103px;">Life Technologies</td>
			<td style="width:119px;">F-150</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:216px;">Magnetic Stand</td>
			<td style="width:103px;">Promega</td>
			<td style="width:119px;">Z5332</td>
			<td></td>
		</tr>
	</tbody>
</table>

antibody labeling with fluorescent dyes using magnetic protein a and protein g beads

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, immunodiagnostics and more recently as therapeutic agents. Traditional methods for labeling antibodies with small molecules require purified antibodies at relatively high concentration, involve multiple dialysis steps and have limited throughput. However, several applications, including the field of Antibody Drug Conjugates (ADCs), will benefit from new methods that will allow labeling of antibodies directly from cell media. Such methods may allow antibodies to be screened in biologically relevant assays, for example, the receptor-mediated antibody internalization assay in the case of ADCs. Here, we describe a method (on-bead method) that enables labeling of small amounts of antibodies directly from cell media. This approach utilizes high capacity magnetic Protein A and Protein G affinity beads to capture antibodies from the cell media followed by labeling with small molecules using either amine or thiol chemistry and subsequent elution of the labeled antibodies. Taking fluorescent dyes as surrogates for small molecules, we demonstrate the on-bead labeling of three different mouse antibodies directly from cell media using both amine and thiol labeling chemistry. The high binding affinity of antibodies to Protein A and Protein G ensures high recoveries as well as high purity of the labeled antibodies. In addition, use of magnetic beads allows multiple samples to be handled manually, thereby significantly improving labeling throughput.

A schematic for labeling antibodies with small molecules using high capacity magnetic Protein A and Protein G beads is shown in Figure 1. Antibodies captured on the magnetic Protein G beads can be labeled with small molecules, for example fluorescent dyes, using either amine chemistry which labels primary amines of the lysine amino acids or using thiol chemistry which labels at the reduced thiols in the hinge region of the antibodies. Affinity between antibodies and Prote...

Watch this Scientific Journal Video about Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads at JoVE.com

Antibody Labeling with Fluorescent Dyes Using Magnetic Protein A and Protein G Beads

The on-bead method for labeling antibodies with small molecules enables labeling of a small amount of antibodies directly from cell media. This method is compatible with amine and thiol chemistry, and can handle multiple samples in parallel, manually or using automated platforms.

Antibodies labeled with small molecules like fluorescent dyes, cytotoxic drugs, and radioactive tracers are essential tools in biomedical research, ...

Research

JoVE Journal

Immunology and Infection

Specific Marking of HIV-1 Positive Cells using a Rev-dependent Lentiviral Vector Expressing the Green Fluorescent Protein

Most of HIV-responsive expression vectors are based on the HIV promoter, the long terminal repeat (LTR). While responsive to an early HIV protein, Tat, ...

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito (Aedes aegypti) and Tick (Rhipicephalus microplus) G Protein-coupled Receptors

A Calcium Bioluminescence Assay for Functional Analysis of Mosquito Aedes aegypti and Tick Rhipicephalus microplus G Protein-coupled Receptors

Arthropod hormone receptors are potential targets for novel pesticides as they regulate many essential physiological and behavioral processes.  The ...

Colorectal Cancer Cell Surface Protein Profiling Using an Antibody Microarray and Fluorescence Multiplexing

The current prognosis and classification of CRC relies on staging systems that integrate histopathologic and clinical findings. However, in the majority ...

Protein Misfolding Cyclic Amplification of Prions

Prions  are infectious agents that cause the inevitably fatal transmissible spongiform  encephalopathy (TSE) in animals and humans9,18. The prion protein ...

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Monitoring Changes in Membrane Polarity, Membrane Integrity, and Intracellular Ion Concentrations in Streptococcus pneumoniae Using Fluorescent Dyes

Membrane depolarization and ion fluxes are events that have been studied extensively in biological systems due to their ability to profoundly impact ...

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

Inoculating Anopheles gambiae Mosquitoes with Beads to Induce and Measure the Melanization Immune Response

The stimulation of immune responses is a common tool in invertebrate studies to examine the efficacy and the mechanisms of immunity. This stimulation is ...

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

A Simple Flow Cytometry Based Assay to Determine In Vitro Antibody Dependent Enhancement of Dengue Virus Using Zika Virus Convalescent Serum

Antibody dependent enhancement of infection has been shown to play a major role in Dengue viral pathogenesis. Traditional assays that measure the capacity ...

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

A Protein Microarray Assay for Serological Determination of Antigen-specific Antibody Responses Following Clostridium difficile Infection

We provide a detailed overview of a novel high-throughput protein microarray assay for the determination of anti-Clostridium difficile antibody levels in ...