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Affinity chromatography

Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another...

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Affinity chromatography is a method of separating a biomolecule from a mixture, based on a highly specific macromolecular binding interaction between the biomolecule and another substance. The specific type of binding interaction depends on the biomolecule of interest; antigen and antibody, enzyme and substrate, receptor and ligand, or protein and nucleic acid[1] binding interactions are frequently exploited for isolation of various biomolecules. Affinity chromatography is useful for its high selectivity and resolution of separation,[2][3] compared to other chromatographic methods.

Principle

Affinity chromatography has the advantage of specific binding interactions between the analyte of interest (normally dissolved in the mobile phase), and a binding partner or ligand (immobilized on the stationary phase). In a typical affinity chromatography experiment, the ligand is attached to a solid, insoluble matrix—usually a polymer such as agarose or polyacrylamide—chemically modified to introduce reactive functional groups with which the ligand can react, forming stable covalent bonds.[4] The stationary phase is first loaded into a column to which the mobile phase is introduced. Molecules that bind to the ligand will remain associated with the stationary phase. A wash buffer is then applied to remove non-target biomolecules by disrupting their weaker interactions with the stationary phase, while the biomolecules of interest will remain bound. Target biomolecules may then be removed by applying a so-called elution buffer, which disrupts interactions between the bound target biomolecules and the ligand. The target molecule is thus recovered in the eluting solution.[5]

Affinity chromatography does not require the molecular weight, charge, hydrophobicity, or other physical properties of the analyte of interest to be known, although knowledge of its binding properties is useful in the design of a separation protocol.[5] Types of binding interactions commonly exploited in affinity chromatography procedures are summarized in the table below.

Batch and column setups

Principle of affinity column chromatography
Batch chromatography

Binding to the solid phase may be achieved by column chromatography whereby the solid medium is packed onto a column, the initial mixture run through the column to allow settling, a wash buffer run through the column and the elution buffer subsequently applied to the column and collected. These steps are usually done at ambient pressure. Alternatively, binding may be achieved using a batch treatment, for example, by adding the initial mixture to the solid phase in a vessel, mixing, separating the solid phase, removing the liquid phase, washing, re-centrifuging, adding the elution buffer, re-centrifuging and removing the elute.

Sometimes a hybrid method is employed such that the binding is done by the batch method, but the solid phase with the target molecule bound is packed onto a column and washing and elution are done on the column.

The ligands used in affinity chromatography are obtained from both organic and inorganic sources. Examples of biological sources are serum proteins, lectins and antibodies. Inorganic sources are moronic acid, metal chelates and triazine dyes.[7]

A third method, expanded bed absorption, which combines the advantages of the two methods mentioned above, has also been developed. The solid phase particles are placed in a column where liquid phase is pumped in from the bottom and exits at the top. The gravity of the particles ensure that the solid phase does not exit the column with the liquid phase.

Affinity columns can be eluted by changing salt concentrations, pH, pI, charge and ionic strength directly or through a gradient to resolve the particles of interest.

More recently, setups employing more than one column in series have been developed. The advantage compared to single column setups is that the resin material can be fully loaded since non-binding product is directly passed on to a consecutive column with fresh column material. These chromatographic processes are known as periodic counter-current chromatography (PCC). The resin costs per amount of produced product can thus be drastically reduced. Since one column can always be eluted and regenerated while the other column is loaded, already two columns are sufficient to make full use of the advantages.[8] Additional columns can give additional flexibility for elution and regeneration times, at the cost of additional equipment and resin costs.

Specific uses

Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification[9] from cell free extracts, and purification from blood.

By using affinity chromatography, one can separate proteins that bind to a certain fragment from proteins that do not bind that specific fragment.[10] Because this technique of purification relies on the biological properties of the protein needed, it is a useful technique and proteins can be purified many folds in one step.[11]

Various affinity media

Many different affinity media exist for a variety of possible uses.[12][9][13] Briefly, they are (generalized) activated/functionalized that work as a functional spacer, support matrix, and eliminates handling of toxic reagents.

Amino acid media is used with a variety of serum proteins, proteins, peptides, and enzymes, as well as rRNA and dsDNA. Avidin biotin media is used in the purification process of biotin/avidin and their derivatives.

Carbohydrate bonding is most often used with glycoproteins or any other carbohydrate-containing substance; carbohydrate is used with lectins, glycoproteins, or any other carbohydrate metabolite protein. Dye ligand media is nonspecific but mimics biological substrates and proteins. Glutathione is useful for separation of GST tagged recombinant proteins. Heparin is a generalized affinity ligand, and it is most useful for separation of plasma coagulation proteins, along with nucleic acid enzymes and lipases

Hydrophobic interaction media are most commonly used to target free carboxyl groups and proteins.

Immunoaffinity media (detailed below) utilizes antigens' and antibodies' high specificity to separate; immobilized metal affinity chromatography is detailed further below and uses interactions between metal ions and proteins (usually specially tagged) to separate; nucleotide/coenzyme that works to separate dehydrogenases, kinases, and transaminases.

Nucleic acids function to trap mRNA, DNA, rRNA, and other nucleic acids/oligonucleotides. Protein A/G method is used to purify immunoglobulins.

Speciality media are designed for a specific class or type of protein/co enzyme; this type of media will only work to separate a specific protein or coenzyme.

Immunoaffinity

Otro uso de este procedimiento es la purificación por afinidad de anticuerpos a partir de suero sanguíneo. Si se sabe que el suero contiene anticuerpos contra un antígeno específico (por ejemplo, si proviene de un organismo inmunizado contra dicho antígeno), puede utilizarse para la purificación por afinidad de ese antígeno. Esto también se conoce como cromatografía de inmunoafinidad. Por ejemplo, si un organismo se inmuniza contra una proteína de fusión GST, producirá anticuerpos contra la proteína de fusión y, posiblemente, también contra la etiqueta GST. La proteína puede entonces unirse covalentemente a un soporte sólido, como la agarosa, y utilizarse como ligando de afinidad en la purificación de anticuerpos a partir de suero inmune.

Para mayor precisión, la proteína GST y la proteína de fusión GST pueden acoplarse por separado. Inicialmente, se permite que el suero se una a la matriz de afinidad GST. Esto eliminará los anticuerpos contra la parte GST de la proteína de fusión. A continuación, el suero se separa del soporte sólido y se deja que se una a la matriz de la proteína de fusión GST. Esto permite que cualquier anticuerpo que reconozca el antígeno quede capturado en el soporte sólido. La elución de los anticuerpos de interés se suele lograr utilizando un tampón de pH bajo , como glicina pH 2,8. El eluato se recoge en un tampón de fosfato o tris neutro para neutralizar el tampón de elución de pH bajo y detener cualquier degradación de la actividad del anticuerpo. Este es un buen ejemplo, ya que la purificación por afinidad se utiliza para purificar la proteína de fusión GST inicial, eliminar los anticuerpos anti-GST no deseados del suero y purificar el anticuerpo objetivo.

También se pueden seleccionar anticuerpos monoclonales para que se unan a proteínas con gran especificidad, donde la proteína se libera en condiciones relativamente suaves. Esto puede resultar útil para futuras investigaciones. [ 14 ]

Se suele emplear una estrategia simplificada para purificar anticuerpos generados contra antígenos peptídicos . Cuando los antígenos peptídicos se sintetizan, se añade un residuo de cisteína terminal en el extremo N o C del péptido. Este residuo de cisteína contiene un grupo funcional sulfhidrilo que permite que el péptido se conjugue fácilmente a una proteína portadora (por ejemplo, hemocianina de lapa (KLH)). El mismo péptido con cisteína se inmoviliza en una resina de agarosa a través del residuo de cisteína y se utiliza posteriormente para purificar el anticuerpo.

La mayoría de los anticuerpos monoclonales se han purificado utilizando cromatografía de afinidad basada en la proteína A o la proteína G específicas de inmunoglobulina , derivadas de bacterias. [ 15 ]

Immunoaffinity chromatography with monoclonal antibodies immobilized on monolithic column has been successfully used to capture extracellular vesicles (e.g., exosomes and exomeres) from human blood plasma by targeting tetraspanins and integrins found on the surface of the EVs.[16][17]

Immunoaffinity chromatography is also the basis for immunochromatographic test (ICT) strips, which provide a rapid means of diagnosis in patient care. Using ICT, a technician can make a determination at a patient's bedside, without the need for a laboratory.[18] ICT detection is highly specific to the microbe causing an infection.[19]

Immunoaffinity chromatography has been applied to the selective isolation of blood-derived components, including lymphocytes and specific proteins, using antibody-functionalized stationary phases.[20] Although not a routine method for estimating the time since deposition (TSD) of bloodstains, targeted molecular analyses used in TSD research – such as proteomics, metabolomics, and nucleic-acid profiling – may incorporate immunoaffinity-based enrichment steps to improve sensitivity for low-abundance biomarkers.[21][22] Antibody-based detection methods are also used in forensic serology to identify blood and evaluate its persistence on substrates over time, demonstrating the broader role of immunological specificity in forensic body-fluid analysis.[23]

Immobilized metal ion affinity chromatography

Immobilized metal ion affinity chromatography (IMAC) is based on the specific coordinate covalent bond of amino acids, particularly histidine, to metals. This technique works by allowing proteins with an affinity for metal ions to be retained in a column containing immobilized metal ions, such as cobalt, nickel, or copper for the purification of histidine-containing proteins or peptides, iron, zinc or gallium for the purification of phosphorylated proteins or peptides. Many naturally occurring proteins do not have an affinity for metal ions, therefore recombinant DNA technology can be used to introduce such a protein tag into the relevant gene. Methods used to elute the protein of interest include changing the pH, or adding a competitive molecule, such as imidazole.[24][25]

A chromatography column containing nickel-agarose beads used for purification of proteins with histidine tags

Recombinant proteins

Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Proteins with a known affinity are protein tagged in order to aid their purification. The protein may have been genetically modified so as to allow it to be selected for affinity binding; this is known as a fusion protein. Protein tags include hexahistidine (His), glutathione-S-transferase (GST), maltose binding protein (MBP), and the Colicin E7 variant CL7 tag. Histidine tags have an affinity for nickel, cobalt, zinc, copper and iron ions which have been immobilized by forming coordinate covalent bonds with a chelator incorporated in the stationary phase. For elution, an excess amount of a compound able to act as a metal ion ligand, such as imidazole, is used. GST has an affinity for glutathione which is commercially available immobilized as glutathione agarose. During elution, excess glutathione is used to displace the tagged protein. CL7 has an affinity and specificity for Immunity Protein 7 (Im7) which is commercially available immobilized as Im7 agarose resin. For elution, an active and site-specific protease is applied to the Im7 resin to release the tag-free protein.[26]

Lectins

Lectin affinity chromatography is a form of affinity chromatography where lectins are used to separate components within the sample. Lectins, such as concanavalin A are proteins which can bind specific alpha-D-mannose and alpha-D-glucose carbohydrate molecules. Some common carbohydrate molecules that is used in lectin affinity chromatography are Con A-Sepharose and WGA-agarose.[27] Another example of a lectin is wheat germ agglutinin which binds D-N-acetyl-glucosamine.[28] The most common application is to separate glycoproteins from non-glycosylated proteins, or one glycoform from another glycoform.[29] Although there are various ways to perform lectin affinity chromatography, the goal is extract a sugar ligand of the desired protein.[27]

Specialty

Another use for affinity chromatography is the purification of specific proteins using a gel matrix that is unique to a specific protein. For example, the purification of E. coli β-galactosidase is accomplished by affinity chromatography using p-aminobenyl-1-thio-β-D-galactopyranosyl agarose as the affinity matrix. p-aminobenyl-1-thio-β-D-galactopyranosyl agarose is used as the affinity matrix because it contains a galactopyranosyl group, which serves as a good substrate analog for E. coli β-Galactosidase. This property allows the enzyme to bind to the stationary phase of the affinity matrix and β-Galactosidase is eluted by adding increasing concentrations of salt to the column.[30]

Alkaline phosphatase

Alkaline phosphatase from E. coli can be purified using a DEAE-Cellulose matrix. A. phosphatase has a slight negative charge, allowing it to weakly bind to the positively charged amine groups in the matrix. The enzyme can then be eluted out by adding buffer with higher salt concentrations.[31]

Boronate affinity chromatography

Boronate affinity chromatography consists of using boronic acid or boronates to elute and quantify amounts of glycoproteins. Clinical adaptations have applied this type of chromatography for use in determining long term assessment of diabetic patients through analysis of their glycated hemoglobin.[28]

Serum albumin purification

Affinity purification of albumin and macroglobulin contamination is helpful in removing excess albumin and α2-macroglobulin contamination, when performing mass spectrometry. In affinity purification of serum albumin, the stationary used for collecting or attracting serum proteins can be Cibacron Blue-Sepharose. Then the serum proteins can be eluted from the adsorbent with a buffer containing thiocyanate (SCN−).[32]

Weak affinity chromatography

La cromatografía de afinidad débil [ 33 ] ( WAC ) es una técnica de cromatografía de afinidad para la detección de afinidad en el desarrollo de fármacos. [ 34 ] [ 35 ] La WAC es una técnica de cromatografía líquida basada en la afinidad que separa compuestos químicos según sus diferentes afinidades débiles a una diana inmovilizada. Cuanto mayor sea la afinidad de un compuesto hacia la diana, más tiempo permanecerá en la unidad de separación, lo que se expresará como un mayor tiempo de retención. La medición y clasificación de la afinidad se puede obtener procesando los tiempos de retención de los compuestos analizados. La cromatografía de afinidad forma parte de un conjunto más amplio de técnicas utilizadas en la identificación de dianas farmacológicas basada en quimioproteómica .

La tecnología WAC se ha probado frente a diversos objetivos proteicos : proteasas , quinasas , chaperonas y objetivos de interacción proteína-proteína (PPI). Se ha demostrado que WAC es más eficaz que los métodos establecidos para el cribado basado en fragmentos. [ 35 ]

Historia

La cromatografía de afinidad fue concebida y desarrollada por primera vez por Pedro Cuatrecasas y Meir Wilchek . [ 36 ] [ 37 ]

Referencias

  1. ↑ Aizpurua-Olaizola, Oier; Sastre Torano, Javier; Pukin, Aliaksei; Fu, ou; Bendiciones, Geert Jan; de Jong, Gerhardus J.; Pieters, Roland J. (enero de 2018). "Electroforesis capilar de afinidad para la evaluación de la afinidad de unión de inhibidores de la toxina del cólera a base de carbohidratos". Electroforesis . 39 (2): 344– 347. doi : 10.1002/elps.201700207 . hdl : 1874/362143 . PMID 28905402 . S2CID 33657660 .  
  2. ↑ Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2009). Enfoques fundamentales de laboratorio para bioquímica y biotecnología (2.ª ed.). Wiley. pág. 133. ISBN   978-0-470-08766-4.
  3. ↑ ""Introducción a la cromatografía de afinidad"" . bio-rad.com . Bio-Rad. 14 de septiembre de 2020 . Consultado el 14 de septiembre de 2020 .
  4. ↑ Zachariou, Michael, ed. (2008). Cromatografía de afinidad: métodos y protocolos (2.ª ed.). Totowa, NJ: Humana Press. pp. 1–2 . ISBN   978-1-58829-659-7.
  5. 12Bonner, Philip L.R. (2007). Protein Purification (2nd ed.). Totowa, N.J.: Taylor & Francis Group. ISBN 978-0-415-38511-4.
  6. ↑Kumar, Pranav (2018). Biophysics and Molecular Biology. New Delhi: Pathfinder Publication. p. 11. ISBN 978-93-80473-15-4.
  7. ↑Fanali, Salvatore; Haddad, Paul R.; Poole, Colin F.; Schoenmakers, Peter; Lloyd, David, eds. (2013). Liquid Chromatography: Applications. Handbooks in Separation Science. Saint Louis: Elsevier. p. 3. ISBN 978-0-12-415806-1.
  8. ↑Baur, Daniel; Angarita, Monica; Müller-Späth, Thomas; Steinebach, Fabian; Morbidelli, Massimo (2016). "Comparison of batch and continuous multi-column protein A capture processes by optimal design". Biotechnology Journal. 11 (7): 920–931. doi:10.1002/biot.201500481. hdl:11311/1013726. PMID 26992151. S2CID 205492204.
  9. 12"Cube Biotech". Cube Biotech. Retrieved 11 September 2019.
  10. ↑Ahern, Kevin; Rajagopal, Indira (12 February 2015). Biochemistry Free & Easy (3rd ed.). DaVinci Press. p. 822. hdl:10211.3/206119.
  11. ↑Grisham, Charles M. (1 January 2013). Biochemistry. Brooks/Cole, Cengage Learning. ISBN 978-1-133-10629-6. OCLC 777722371.
  12. ↑Mahmoudi Gomari, Mohammad; Saraygord-Afshari, Neda; Farsimadan, Marziye; Rostami, Neda; Aghamiri, Shahin; Farajollahi, Mohammad M. (1 December 2020). "Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry". Biotechnology Advances. 45 107653. doi:10.1016/j.biotechadv.2020.107653. ISSN 0734-9750. PMID 33157154. S2CID 226276355.
  13. ↑"Affinity Chromatography".
  14. ↑Thompson, Nancy E.; Foley, Katherine M.; Stalder, Elizabeth S.; Burgess, Richard R. (2009). "Chapter 28 Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography". Guide to Protein Purification, 2nd Edition. Methods in Enzymology. Vol. 463. pp. 475–494. doi:10.1016/s0076-6879(09)63028-7. ISBN 978-0-12-374536-1. PMID 19892188.
  15. ↑Uhlén, Mathias (April 2008). "Affinity as a Tool in Life Science". BioTechniques. 44 (5): 649–654. doi:10.2144/000112803. PMID 18474040.
  16. ↑Multia, Evgen; Tear, Crystal Jing Ying; Palviainen, Mari; Siljander, Pia; Riekkola, Marja-Liisa (December 2019). "Fast isolation of highly specific population of platelet-derived extracellular vesicles from blood plasma by affinity monolithic column, immobilized with anti-human CD61 antibody". Analytica Chimica Acta. 1091: 160–168. Bibcode:2019AcAC.1091..160M. doi:10.1016/j.aca.2019.09.022. hdl:10138/321264. PMID 31679569. S2CID 203147714.
  17. ↑Multia, Evgen; Liangsupree, Thanaporn; Jussila, Matti; Ruiz-Jimenez, Jose; Kemell, Marianna; Riekkola, Marja-Liisa (6 October 2020). "Automated On-Line Isolation and Fractionation System for Nanosized Biomacromolecules from Human Plasma". Analytical Chemistry. 92 (19): 13058–13065. doi:10.1021/acs.analchem.0c01986. PMC 7586295. PMID 32893620.
  18. ↑Luppa, Peter (2018). Point-of-care testing: principles and clinical applications. Berlin, Germany: Springer. pp. 71–72. ISBN 978-3-662-54497-6.
  19. ↑Muller, JD; Wilks, CR; O'Riley, KJ; Condron, RJ; Bull, R.; Mateczun, A. (April 2004). "Specificity of an immunochromato-graphic test for anthrax". Australian Veterinary Journal. 82 (4): 220–222. doi:10.1111/j.1751-0813.2004.tb12682.x. PMID 15149073.
  20. ↑Mohr, Fabian; Przibilla, Sabine; Leonhardt, Franziska; Stemberger, Christian; Dreher, Stefan; Müller, Thomas R; Fräßle, Simon P; Schmidt, Georg P; Kiene, Marie-Luise; Stadler, Herbert; Busch, Dirk H (13 November 2018). "Efficient immunoaffinity chromatography of lymphocytes directly from whole blood". Scientific Reports. 8.
  21. ↑Elliott, Colin I; Shafer, Aaron B; Stotesbury, Theresa E (23 October 2022). "The crux of time: A meta-analysis of ex vivo whole blood degradation". Forensic Science International: Synergy. 2.
  22. ↑Sacco, Matteo A; Gualtieri, Saverio; Tarallo, Alessandro P; Calanna, Luca; Russa, Raffaele L; Aquila, Isabella (8 July 2024). "The Role of Molecular Investigations in Estimating the Time since Deposition (TSD) of Bloodstains: A Systematic Review of the Literature"(PDF). Forensic Chemistry. 30.
  23. ↑Medina-Paz, Fransisco; Kuba, Brandon; Kryvorutsky, Emily; Roca, Gabriela; Zapico, Sara C (20 March 2024). "Assessment of Blood and Semen Detection and DNA Collection from Swabs up to Three Months after Deposition on Five Different Cloth Materials". Forensic Science International. 25.
  24. ↑Singh, Naveen K.; DSouza, Roy N.; Bibi, Noor S.; Fernández-Lahore, Marcelo (2015). "Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography". In Reichelt, S. (ed.). Affinity Chromatography. Methods in Molecular Biology. Vol. 1286. New York: Humana Press. pp. 201–212. doi:10.1007/978-1-4939-2447-9_16. ISBN 978-1-4939-2447-9. PMID 25749956.
  25. ↑Gaberc-Porekar, Vladka K.; Menart, Viktor (2001). "Perspectives of immobilized-metal affinity chromatography". J Biochem Biophys Methods. 49 (1–3): 335–360. doi:10.1016/S0165-022X(01)00207-X. PMID 11694288.
  26. ↑ Vassylyeva, Marina N.; Klyuyev, Sergiy; Vassylyev, Alexey D.; Wesson, Hunter; Zhang, Zhuo; Renfrow, Matthew B.; Wang, Hengbin; Higgins, N. Patrick; Chow, Louise T.; Vassylyev, Dmitry G. (27 de junio de 2017). "Cromatografía eficiente de ultra alta afinidad en una purificación en un solo paso de proteínas complejas" . Actas de la Academia Nacional de Ciencias de los Estados Unidos de América . 114 (26): E5138– E5147. Bibcode : 2017PNAS..114E5138V . doi : 10.1073/pnas.1704872114 . ISSN 0027-8424 . PMC 5495267 . PMID 28607052 .   
  27. 1 2 Freeze, HH (mayo de 2001). «Cromatografía de afinidad con lectinas». Current Protocols in Protein Science . págs. 9.1.1–9.1.9. doi : 10.1002/0471140864.ps0901s00 . ISBN  978-0-471-14086-3. ISSN 1934-3663 . PMID 18429210 . S2CID 3197260 .   
  28. 1 2 Hage, David (mayo de 1999). "Cromatografía de afinidad: una revisión de las aplicaciones clínicas" (PDF) . Química clínica . 45 (5): 593– 615. doi : 10.1093/clinchem/45.5.593 . PMID 10222345 . 
  29. ↑ "GE Healthcare Life Sciences, Lectina inmovilizada" . Archivado del original el 3 de marzo de 2012. Consultado el 29 de noviembre de 2010 .
  30. ↑ Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2006). Enfoques fundamentales de laboratorio para bioquímica y biotecnología (2.ª ed.). Wiley. p. 153.  
  31. ↑ Ninfa, Alexander J.; Ballou, David P.; Benore, Marilee (2010). Enfoques fundamentales de laboratorio para bioquímica y biotecnología (2.ª ed.). Hoboken, NJ: John Wiley. p. 240. ISBN   978-0-470-08766-4OCLC 420027217​​ 
  32. ↑ Naval, Javier; Calvo, Miguel; Lampreave, Fermín; Piñeiro, Andrés (1 de enero de 1983). "Cromatografía de afinidad de albúmina sérica: un experimento de laboratorio ilustrativo sobre interacciones biomoleculares". Educación Bioquímica . 11 (1): 5– 8. doi : 10.1016/0307-4412(83)90004-3 . ISSN 1879-1468 . 
  33. ↑ Zopf, D.; Ohlson, S. (julio de 1990). "Cromatografía de baja afinidad". Nature . 346 (6279): 87– 88. Bibcode : 1990Natur.346...87Z . doi : 10.1038/346087a0 . ISSN 0028-0836 . S2CID 4306269 .  
  34. ↑ Duong-Thi, MD; Meiby, E.; Bergström, M.; Fex, T.; Isaksson, R.; Ohlson, S. (2011). "Cromatografía de afinidad débil como un nuevo enfoque para la detección de fragmentos en el descubrimiento de fármacos". Analytical Biochemistry . 414 (1): 138– 146. doi : 10.1016/j.ab.2011.02.022 . PMID 21352794 . 
  35. 1 2 Meiby, E.; Simmonite, H.; Le Strat, L.; Davis, B.; Matassova, N.; Moore, JD; Mrosek, M.; Murray, J.; Hubbard, RE; Ohlson, S. (2013). "Fragment Screening by Weak Affinity Chromatography: Comparison with Established Techniques for Screening against HSP90". Analytical Chemistry . 85 (14): 6756– 6766. doi : 10.1021/ac400715t . PMID 23806099 . 
  36. ↑ "Meir Wilchek - Fundación Wolf" . Fundación Wolf . 9 de diciembre de 2018. Consultado el 17 de marzo de 2021. La cromatografía de afinidad es una técnica novedosa concebida por Cuatrecasas y Wilchek .
  37. ↑ Cuatrecasas, P; Wilchek, M; Anfinsen, CB (octubre de 1968). "Purificación selectiva de enzimas mediante cromatografía de afinidad" . Actas de la Academia Nacional de Ciencias . 61 (2): 636– 643. Bibcode : 1968PNAS...61..636C . doi : 10.1073/pnas.61.2.636 . PMC 225207. PMID 4971842 .  
  • "Cromatografía de afinidad: Principio, procedimiento y nota detallada avanzada – 2020".
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