Name: antibody labeling with fluorescent dyes using magnetic protein a and protein g beads
Uploaded: 2016-09-15T14:00:00.000+00:00
Duration: 6 min 48 s
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 of the sample containing 50-100 &#181;g of antibody to the beads. Samples can be purified antibodies or antibodies in cell media.</li>
	<li>Mix sample for 60 min at RT using a vortex mixer or end-over-end mixer.</li>
	<li>Place tube in the magnetic stand for 10 sec and remove the supernatant.</li>
	<li>Add 250 &#181;l of antibody binding/washing buffer and mix. Place in the magnetic stand for 10 sec and remove binding/washing buffer. Repeat this step for a total of two washes.</li>
	<li>Proceed to Section 2 to label antibodies using amine chemistry or to Section 3 to label antibodies using thiol chemistry.</li>
</ol>
2. Antibody Labeling Using Amine Chemistry
<ol>
	<li>Conjugate Antibody
	<ol>
		<li>Add 100 &#181;l of amine conjugation buffer to the beads.</li>
		<li>Dissolve the amine-reactive fluorescent dyes (AlexaFluor 532-SE) at 10 mg/ml by adding 100 &#181;l of Dimethyl sulfoxide (DMSO) to 1.0 mg of dye. Mix by vortexing. Make this solution just before use.</li>
		<li>Add 2.5 &#181;l of amine-reactive dye for 100 &#181;g of antibody. 
		Note: Typically a 5-20 molar excess of reactive dye is recommended. However, amount of reactive dye added to the reaction needs to be empirically optimized depending on desired dye to antibody ratio and intrinsic antibody properties.</li>
		<li>Mix sample for 60 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.</li>
		<li>Place tube in the magnetic stand for 10 sec and remove the supernatant.</li>
		<li>Add 250 &#181;l of antibody binding/washing buffer and mix. Place in the magnetic stand for 10 sec. Remove and discard binding/washing buffer. Repeat this step for a total of two washes.</li>
	</ol>
	</li>
	<li>Antibody Recovery
	<ol>
		<li>Add 50 &#181;l of elution buffer to the beads.</li>
		<li>Mix for 5 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.</li>
		<li>Place tube in the magnetic stand for 10 sec. Remove eluted sample and transfer to a new micro-centrifuge tube containing 5 &#181;l of neutralization buffer.</li>
		<li>Repeat the process one more time and pool the eluted samples.</li>
		<li>Quantitate the antibody concentration and dye-to-antibody ratio as described in Section 4.</li>
	</ol>
	</li>
</ol>
3. Antibody Labeling Using Thiol Chemistry
<ol>
	<li>Antibody Reduction
	<ol>
		<li>Add 250 &#181;l of thiol conjugation buffer and mix. Place the tube in the magnetic stand for 10 sec. Remove and discard the buffer. Repeat this step twice.</li>
		<li>Add 100 &#181;l of thiol conjugation buffer.</li>
		<li>Add Dithiothreitol (DTT) to a final concentration of 2.5 mM.</li>
		<li>Mix sample for 60 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.</li>
		<li>Place the tube in the magnetic stand for 10 sec and discard the buffer.</li>
		<li>Add 250 &#181;l of thiol conjugation buffer and mix. Place the tube in the magnetic stand for 10 sec. Remove and discard the buffer. Repeat this step for a total of two washes.</li>
		<li>Add 100 &#181;l of thiol conjugation buffer.</li>
	</ol>
	</li>
	<li>Conjugate Antibody
	<ol>
		<li>Dissolve the thiol-reactive dye at 10 mg/ml by adding 100 &#181;l of DMSO to 1.0 mg of dye. Mix by vortexing. Make this solution just before use.</li>
		<li>Add 2.5 &#181;l of thiol-reactive dye for 100 &#181;g of antibody. 
		Note: Typically a 5-20 molar excess of reactive dye is recommended. However, amount of reactive dye added to the reaction needs to be empirically optimized depending on desired dye to antibody ratio and intrinsic antibody properties.</li>
		<li>Mix sample for 60 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.</li>
		<li>Place the tube in the magnetic stand for 10 sec and remove supernatant.</li>
		<li>Add 250 &#181;l of thiol conjugation buffer and mix. Place in the magnetic stand for 10 sec and remove the buffer. Repeat this step for a total of two washes.</li>
	</ol>
	</li>
	<li>Elute Antibody
	<ol>
		<li>Add 50 &#181;l of elution buffer to the beads.</li>
		<li>Mix for 5 min at RT using a vortex mixer or end-over-end mixer. Make sure that the beads remain in suspension.</li>
		<li>Place tube in the magnetic stand for 10 sec. Remove eluted sample and transfer to a new micro-centrifuge tube containing 5 &#181;l of neutralization buffer.</li>
		<li>Repeat the process one more time and pool the eluted samples.</li>
		<li>Quantitate the antibody concentration and dye-to-antibody ratio as described in Section 4.</li>
	</ol>
	</li>
</ol>
4. Calculate Dye-to-Antibody Ratio
<ol>
	<li>Measure the absorbance of the antibody-dye conjugate at 280 nm (A280) and at the &#955;max for the dye (Amax).</li>
	<li>Calculate the antibody concentration: 
	Antibody Concentration (mg/ml) = A280 - (Amax &#215; CF) / 1.4 
	where CF = Correction factor of the dye (provided by the manufacturer).</li>
	<li>Calculate the dye-to-antibody ratio: 
	Dye-to-Antibody Ratio (DAR) = (Amax &#215; 150,000)/Ab Concentration (mg/ml) &#215; &#949;dye 
	where, &#949;dye = the extinction coefficient of the dye at its absorbance maximum and molecular weight of antibody = 150,000 Da.</li>
</ol>

<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>

Authors are employees of Promega Corporation.

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><tbody><tr><td>Magne Protein A Beads</td><td>Promega Corporation</td><td>&amp;nbsp;G8781</td><td></td></tr><tr><td>Magne Protein G Beads</td><td>Promega Corporation&amp;nbsp;</td><td>&amp;nbsp;G7471</td><td></td></tr><tr><td>AlexaFluor 532-SE (Succinimidyl Ester)</td><td>Life Technologies</td><td>A20001</td><td></td></tr><tr><td>AlexaFluor 532-ME (Malemide)</td><td>Life Technologies</td><td>A10255</td><td></td></tr><tr><td>AlexaFluor 647-ME (Maleimide)</td><td>Life Technologies</td><td>A20347</td><td></td></tr><tr><td>Fluorescein-ME (Maleimide)</td><td>Life Technologies</td><td>F-150</td><td></td></tr><tr><td>Magnetic Stand</td><td>Promega</td><td>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, ...

antibody-labeling-with-fluorescent-dyes-using-magnetic-protein

Research

JoVE Journal

Immunology and Infection

11.8K Views.  Promega Corporation. 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.

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

Efficient and Site-specific Antibody Labeling by Strain-promoted Azide-alkyne Cycloaddition

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is protein conjugation by genetically introducing an unnatural amino acid containing a bioorthogonal functional group. This report describes a detailed protocol for site-specific antibody conjugation. The protocol includes experimental details for the genetic incorporation of an azide-containing amino acid, and the conjugation reaction by strain-promoted azide-alkyne cycloaddition (SPAAC). This strain-promoted reaction proceeds by simple mixing of the reacting molecules at physiological pH and temperature, and does not require additional reagents such as copper(I) ions and copper-chelating ligands. Therefore, this method would be useful for general protein conjugation and development of antibody drug conjugates (ADCs).

Here, we present a protocol to site-specifically introduce chemical probes into an antibody fragment by genetically incorporating an azide-containing amino acid, and subsequently coupling the azide with a chemical probe by strain-promoted azide-alkyne cycloaddition (SPAAC).

There are currently many chemical tools available to introduce chemical probes into proteins to study their structure and function. A useful method is ...

Biochemistry

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has relevant health and economic consequences. Many cyanobacterial strains are toxin producers, and only a few cells are necessary to induce irreparable damage to the environment. Therefore, water-body authorities and administrations require rapid and efficient early-warning systems providing reliable data to support their preventive or curative decisions. This manuscript reports an experimental protocol for the in-field detection of toxin-producing cyanobacterial strains by using an antibody microarray chip with 17 antibodies (Abs) with taxonomic resolution (CYANOCHIP). Here, a multiplex fluorescent sandwich microarray immunoassay (FSMI) for the simultaneous monitoring of 17 cyanobacterial strains frequently found blooming in freshwater ecosystems, some of them toxin producers, is described. A microarray with multiple identical replicates (up to 24) of the CYANOCHIP was printed onto a single microscope slide to simultaneously test a similar number of samples. Liquid samples can be tested either by direct incubation with the antibodies (Abs) or after cell concentration by filtration through a 1- to 3-&#956;m filter. Solid samples, such as sediments and ground rocks, are first homogenized and dispersed by a hand-held ultrasonicator in an incubation buffer. They are then filtered (5 - 20 &#956;m) to remove the coarse material, and the filtrate is incubated with Abs. Immunoreactions are revealed by a final incubation with a mixture of the 17 fluorescence-labeled Abs and are read by a portable fluorescence detector. The whole process takes around 3 h, most of it corresponding to two 1-h periods of incubation. The output is an image, where bright spots correspond to the positive detection of cyanobacterial markers.

Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip

The presence of cyanobacterial toxins in fresh water reservoirs for human consumption is a major concern for water management authorities. To evaluate the risk of water contamination, this article describes an protocol for the in-field detection of cyanobacterial strains in liquid and solid samples by using an antibody microarray chip.

Global warming and eutrophication make some aquatic ecosystems behave as true bioreactors that trigger rapid and massive cyanobacterial growth; this has ...

Environment

Differential Labeling of Cell-surface and Internalized Proteins after Antibody Feeding of Live Cultured Neurons

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of live neurons with a specific primary antibody raised against an extracellular portion of the protein. Given that receptors are trafficked to and from the surface, if cells are permeabilized after fixation then both cell-surface and internal protein will be detected by the same labeled secondary antibody. Here, we adapted a method used to study protein trafficking (&#8220;antibody feeding&#8221;) to differentially label protein that had been internalized by endocytosis during the antibody incubation step and protein that either remained on the cell surface or was trafficked to the surface during this period. The ability to distinguish these two pools of protein was made possible through the incorporation of an overnight blocking step with highly-concentrated unlabeled secondary antibody after an initial incubation of unpermeabilized neurons with a fluorescently-labeled secondary antibody. After the blocking step, permeabilization of the neurons allowed detection of the internalized pool with a fluorescent secondary antibody labeled with a different fluorophore. Using this technique we were able to obtain important information about the subcellular location of this putative receptor, revealing that it was, indeed, trafficked to the cell-surface in neurons. This technique is broadly applicable to a range of cell types and cell-surface proteins, providing a suitable antibody to an extracellular epitope is available.

We describe a method to label protein on the surface of living neurons using a specific polyclonal antibody to extracellular epitopes. Protein bound by the antibody on the cell surface and subsequently internalized via endocytosis can be distinguished from protein remaining on, or trafficked to, the surface during the incubation.

In order to demonstrate the cell-surface localization of a putative transmembrane receptor in cultured neurons, we labeled the protein on the surface of ...

Neuroscience

Magnetic Tweezers in a Microplate Format

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these approaches are limited single-molecule methods, which restricts their availability. To address this need, a microplate assay was developed that enables mechanical manipulation while performing standard biochemical assays. This is achieved using magnets incorporated into a microplate lid to create multiple magnetic tweezers. In this format, force is exerted across biomolecules connected to paramagnetic beads, equivalent to a typical magnetic tweezer. The study demonstrates the application of this tool with FRET-based assays to monitor protein conformations. However, this approach is widely applicable to different biological systems ranging from measuring enzymatic activity through to the activation of signaling pathways in live cells.

Here, we describe the use of a novel microplate assay to enable mechanical manipulation of biomolecules while performing ensemble biochemical assays. This is achieved using a microplate lid modified with magnets to create multiple static magnetic tweezers across the microplate.

Mechanobiology describes how the physical forces and mechanical properties of biological material contribute to physiology and disease. Typically, these ...

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude stringent methods for the isolation and identification of protein networks around a protein of interest. The use of chemical crosslinkers allows the selective stabilization of labile interactions, thus bypassing biochemical limitations for purification. Here we present a protocol amenable for cells in culture that uses a homobifunctional crosslinker with a spacer arm of 12 &Aring;, dithiobis-(succinimidyl proprionate) (DSP). DSP is cleaved by reduction of a disulphide bond present in the molecule. Cross-linking combined with immunoaffinity chromatography of proteins of interest with magnetic beads allows the isolation of protein complexes that otherwise would not withstand purification. This protocol is compatible with regular western blot techniques and it can be scaled up for protein identification by mass spectrometry1.
Stephanie A. Zlatic and Pearl V. Ryder contributed equally to this work.

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

The cell permeable crosslinker DSP [dithiobis-(succinimidyl propionate)] stabilizes transient and labile interactions in vivo, which allows their isolation using stringent protein complex purification techniques. Here we present a technique for crosslinking cells grown in culture followed by isolation of protein complexes by immunoprecipitation.

The dynamic nature of cellular machineries is frequently built on transient and/or weak protein associations. These low affinity interactions preclude ...

Biology

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ribonucleoprotein complexes (miRNPs) that are often expressed in a cell-type dependently. To understand how the interplay of these RNA-binding factors affects the regulation of individual transcripts, high resolution maps of in vivo protein-RNA interactions are necessary1.
A combination of genetic, biochemical and computational approaches are typically applied to identify RNA-RBP or RNA-RNP interactions. Microarray profiling of RNAs associated with immunopurified RBPs (RIP-Chip)2 defines targets at a transcriptome level, but its application is limited to the characterization of kinetically stable interactions and only in rare cases3,4 allows to identify the RBP recognition element (RRE) within the long target RNA. More direct RBP target site information is obtained by combining in vivo UV crosslinking5,6 with immunoprecipitation7-9 followed by the isolation of crosslinked RNA segments and cDNA sequencing (CLIP)10. CLIP was used to identify targets of a number of RBPs11-17. However, CLIP is limited by the low efficiency of UV 254 nm RNA-protein crosslinking, and the location of the crosslink is not readily identifiable within the sequenced crosslinked fragments, making it difficult to separate UV-crosslinked target RNA segments from background non-crosslinked RNA fragments also present in the sample.
We developed a powerful cell-based crosslinking approach to determine at high resolution and transcriptome-wide the binding sites of cellular RBPs and miRNPs that we term PAR-CliP (Photoactivatable-Ribonucleoside-Enhanced Crosslinking and Immunoprecipitation) (see Fig. 1A for an outline of the method). The method relies on the incorporation of photoreactive ribonucleoside analogs, such as 4-thiouridine (4-SU) and 6-thioguanosine (6-SG) into nascent RNA transcripts by living cells. Irradiation of the cells by UV light of 365 nm induces efficient crosslinking of photoreactive nucleoside-labeled cellular RNAs to interacting RBPs. Immunoprecipitation of the RBP of interest is followed by isolation of the crosslinked and coimmunoprecipitated RNA. The isolated RNA is converted into a cDNA library and deep sequenced using Solexa technology. One characteristic feature of cDNA libraries prepared by PAR-CliP is that the precise position of crosslinking can be identified by mutations residing in the sequenced cDNA. When using 4-SU, crosslinked sequences thymidine to cytidine transition, whereas using 6-SG results in guanosine to adenosine mutations. The presence of the mutations in crosslinked sequences makes it possible to separate them from the background of sequences derived from abundant cellular RNAs.
Application of the method to a number of diverse RNA binding proteins was reported in Hafner et al.18

RNA transcripts are subject to extensive posttranscriptional regulation that is mediated by a multitude of trans-acting RNA-binding proteins (RBPs). Here we present a generalizable method to identify precisely and on a transcriptome-wide scale the RNA binding sites of RBPs.

RNA transcripts are subjected to post-transcriptional gene regulation by interacting with hundreds of RNA-binding proteins (RBPs) and microRNA-containing ...

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in cell biology. Many phenomena cannot be explained by in vitro analysis in simplified systems and need additional structural information in situ, particularly in the range between 1 nm and 0.1 &#181;m, in order to be fully understood. Here, electron spectroscopic imaging, a transmission electron microscopy technique that allows simultaneous mapping of the distribution of proteins and nucleic acids, and an expression tag, miniSOG, are combined to study the structure and organization of DNA double-strand break repair foci.

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging ESI

Electron spectroscopic imaging can image and distinguish nucleic acid from protein at nanometer resolution. It can be combined with the miniSOG system, which is able to specifically label tagged proteins in transmission electron microscopy samples. We illustrate the use of these technologies using double-strand break repair foci as an example.

The limits to optical resolution and the challenge of identifying specific protein populations in transmission electron microscopy have been obstacles in ...

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A deeper understanding of the changes in gene expression that occur during repopulation could help identify therapeutic targets to promote the restoration of liver function in the setting of injuries. Nonetheless, methods to isolate specifically the repopulating hepatocytes are inhibited by a lack of cell markers, limited cell numbers, and the fragility of these cells. The development of translating ribosome affinity purification (TRAP) technology in conjunction with the Fah-/- mouse model to recapitulate repopulation in the setting of liver injury allows gene expression profiling of the repopulating hepatocytes. With TRAP, cell type-specific translating mRNA is rapidly and efficiently isolated. We developed a method that utilizes TRAP with affinity-based isolation of translating mRNA from hepatocytes that selectively express the green fluorescent protein (GFP)-tagged ribosomal protein (RP), GFP:RPL10A. TRAP circumvents the long time period required for fluorescence-activated cell sorting that could change the gene expression profile. Furthermore, since only the repopulating hepatocytes express the GFP:RPL10A fusion protein, the isolated mRNA is devoid of contamination from the surrounding injured hepatocytes and other cell types in the liver. The affinity-purified mRNA is of high quality and allows downstream PCR- or high-throughput sequencing-based analysis of gene expression.

Translating ribosome affinity purification (TRAP) enables rapid and efficient isolation of cell type-specific translating mRNA. Here, we demonstrate a method that combines hydrodynamic tail-vein injection in a mouse model of liver repopulation and TRAP to examine the expression profile of repopulating hepatocytes.

Liver repopulation after injury is a crucial feature of mammals which prevents immediate organ failure and death after exposure of environmental toxins. A ...

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's disease (PD), Huntington's disease (HD), and so on. To detect and analyze soluble or diffuse protein oligomers or aggregates, fluorescence correlation spectroscopy (FCS), which can detect the diffusion speed and brightness of a single particle with a single molecule sensitivity, has been used. However, the proper procedure and know-how for protein aggregation detection have not been widely shared. Here, we show a standard procedure of FCS measurement for diffusion properties of aggregation-prone proteins in cell lysate and live cells: ALS-associated 25 kDa carboxyl-terminal fragment of TAR DNA/RNA-binding protein 43 kDa (TDP25) and superoxide dismutase 1 (SOD1). The representative results show that a part of aggregates of green fluorescent protein (GFP)-tagged TDP25 was slightly included in the soluble fraction of murine neuroblastoma Neuro2a cell lysate. Moreover, GFP-tagged SOD1 carrying ALS-associated mutation shows a slower diffusion in live cells. Accordingly, we here introduce the procedure to detect the protein aggregation via its diffusion property using FCS.

We here introduce a procedure to measure protein oligomers and aggregation in cell lysate and live cells using fluorescence correlation spectroscopy.

Protein aggregation is a hallmark of neurodegenerative disorders such as amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), Parkinson's ...

A Technique to Detect Cyanobacterial Strains Using an Antibody Microarray Chip

Source: Blanco, Y. et al., <a href="https://app.jove.com/v/54994">Experimental Protocol for Detecting Cyanobacteria in Liquid and Solid Samples with an Antibody Microarray Chip.</a>&#160;J. Vis. Exp. (2017)
This video demonstrates a method to detect the Cyanobacterial strains in an environmental sample using an antibody microarray chip. Samples carrying various bacteria interact with printed antibodies, and immunostaining with fluorophore-labeled anti-cyanobacterial-strain antibodies reveals the presence of specific cyanobacterial strains through fluorescence in distinct spots.

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

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Magnetic Tweezers in a Microplate Format

Isolation of Labile Multi-protein Complexes by in vivo Controlled Cellular Cross-Linking and Immuno-magnetic Affinity Chromatography

PAR-CliP - A Method to Identify Transcriptome-wide the Binding Sites of RNA Binding Proteins

Visualization of miniSOG Tagged DNA Repair Proteins in Combination with Electron Spectroscopic Imaging (ESI)

Cell Type-specific Gene Expression Profiling in the Mouse Liver

Detection of Protein Aggregation using Fluorescence Correlation Spectroscopy

Technika wykrywania szczepów sinic za pomocą chipa mikromacierzy przeciwciał