Editing Affinity chromatography (section)

==Specific uses==
Affinity chromatography can be used in a number of applications, including nucleic acid purification, protein purification<ref name="Cube Biotech">{{Cite web|url=https://cube-biotech.com/affinity-chromatography-which-tag-to-use|title=Cube Biotech|website=Cube Biotech|language=en-GB|access-date=2019-09-11}}</ref> 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.<ref>{{Cite book |last1=Ahern |first1=Kevin |title=Biochemistry Free & Easy |last2=Rajagopal |first2=Indira |date=February 12, 2015 |publisher=DaVinci Press |edition=3rd |page=822 |hdl=10211.3/206119|hdl-access=free}}</ref> 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.<ref>{{Cite book|title=Biochemistry|last=Grisham |first=Charles M. |date=2013-01-01|publisher=Brooks/Cole, Cengage Learning|isbn=978-1133106296|oclc=777722371}}</ref>{{page needed|date=December 2021}}

===Various affinity media===
Many different affinity media exist for a variety of possible uses.<ref>{{cite journal |last1=Mahmoudi Gomari |first1=Mohammad |last2=Saraygord-Afshari |first2=Neda |last3=Farsimadan |first3=Marziye |last4=Rostami |first4=Neda |last5=Aghamiri |first5=Shahin |last6=Farajollahi |first6=Mohammad M. |title=Opportunities and challenges of the tag-assisted protein purification techniques: Applications in the pharmaceutical industry |journal=Biotechnology Advances |date=1 December 2020 |volume=45 |page=107653 |doi=10.1016/j.biotechadv.2020.107653 |pmid=33157154 |s2cid=226276355 |url=https://www.sciencedirect.com/science/article/abs/pii/S0734975020301555 |language=en |issn=0734-9750|url-access=subscription }}</ref><ref name="Cube Biotech"/><ref>{{cite web|url=http://www.sigmaaldrich.com/life-science/proteomics/protein-chromatography/affinity-chromatography.html|title=Affinity Chromatography}}</ref>
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 affinity chromatography|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===
Another use for the procedure is the affinity purification of antibodies from blood serum. If the serum is known to contain antibodies against a specific antigen (for example if the serum comes from an organism immunized against the antigen concerned) then it can be used for the affinity purification of that antigen. This is also known as Immunoaffinity Chromatography. For example, if an organism is immunised against a GST-fusion protein it will produce antibodies against the fusion-protein, and possibly antibodies against the GST tag as well. The protein can then be covalently coupled to a solid support such as agarose and used as an affinity ligand in purifications of antibody from immune serum.

For thoroughness, the GST protein and the GST-fusion protein can each be coupled separately. The serum is initially allowed to bind to the GST affinity matrix. This will remove antibodies against the GST part of the fusion protein. The serum is then separated from the solid support and allowed to bind to the GST-fusion protein matrix. This allows any antibodies that recognize the antigen to be captured on the solid support. Elution of the antibodies of interest is most often achieved using a low [[pH]] buffer such as glycine pH 2.8. The eluate is collected into a neutral [[tris]] or phosphate buffer, to neutralize the low pH elution buffer and halt any degradation of the antibody's activity. This is a nice example as affinity purification is used to purify the initial GST-fusion protein, to remove the undesirable anti-GST antibodies from the serum and to purify the target antibody.

Monoclonal antibodies can also be selected to bind proteins with great specificity, where protein is released under fairly gentle conditions. This can become of use for further research in the future.<ref>{{Cite book|last1=Thompson|first1=Nancy E.|last2=Foley|first2=Katherine M.|last3=Stalder|first3=Elizabeth S.|last4=Burgess|first4=Richard R.|chapter=Chapter 28 Identification, Production, and Use of Polyol-Responsive Monoclonal Antibodies for Immunoaffinity Chromatography |pages=475–494|language=en|doi=10.1016/s0076-6879(09)63028-7|pmid=19892188|title=Guide to Protein Purification, 2nd Edition|volume=463|series=Methods in Enzymology|year=2009|isbn=9780123745361}}</ref>

A simplified strategy is often employed to purify antibodies generated against peptide [[antigen]]s. When the peptide antigens are produced synthetically, a terminal [[cysteine]] residue is added at either the N- or C-terminus of the peptide. This cysteine residue contains a [[sulfhydryl]] functional group which allows the peptide to be easily conjugated to a carrier protein (e.g. Keyhole limpet hemocyanin (KLH)). The same cysteine-containing peptide is also immobilized onto an agarose resin through the cysteine residue and is then used to purify the antibody.

Most [[monoclonal antibody|monoclonal antibodies]] have been purified using affinity chromatography based on [[immunoglobulin]]-specific [[Protein A]] or [[Protein G]], derived from bacteria.<ref name="Uhlen 2008">{{cite journal |last1=Uhlén |first1=Mathias |title=Affinity as a Tool in Life Science |journal=BioTechniques |date=April 2008 |volume=44 |issue=5 |pages=649–654 |doi=10.2144/000112803 | pmid=18474040 | doi-access=free }}</ref>

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.<ref name="Multia et al 2019">{{cite journal |last1=Multia |first1=Evgen |last2=Tear |first2=Crystal Jing Ying |last3=Palviainen |first3=Mari |last4=Siljander |first4=Pia |last5=Riekkola |first5=Marja-Liisa |title=Fast isolation of highly specific population of platelet-derived extracellular vesicles from blood plasma by affinity monolithic column, immobilized with anti-human CD61 antibody |journal=Analytica Chimica Acta |date=December 2019 |volume=1091 |pages=160–168 |doi=10.1016/j.aca.2019.09.022 |pmid=31679569  |bibcode=2019AcAC.1091..160M |hdl=10138/321264 |s2cid=203147714 |hdl-access=free}}</ref><ref name="Multia et al 2020">{{cite journal |last1=Multia |first1=Evgen |last2=Liangsupree |first2=Thanaporn |last3=Jussila |first3=Matti |last4=Ruiz-Jimenez |first4=Jose |last5=Kemell |first5=Marianna |last6=Riekkola |first6=Marja-Liisa |title=Automated On-Line Isolation and Fractionation System for Nanosized Biomacromolecules from Human Plasma |journal=Analytical Chemistry |date=6 October 2020 |volume=92 |issue=19 |pages=13058–13065 |doi=10.1021/acs.analchem.0c01986 |pmid=32893620 |pmc=7586295 |doi-access=free}}</ref>

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.<ref>{{cite book | last=Luppa | first=Peter | title=Point-of-care testing: principles and clinical applications |pages=71–72 | publisher=Springer | location=Berlin, Germany | year=2018 | isbn=9783662544976}}</ref> ICT detection is highly specific to the microbe causing an infection.<ref>{{cite journal |last1=Muller |first1=JD |last2=Wilks |first2=CR |last3=O'Riley |first3=KJ |last4=Condron |first4=RJ |last5=Bull |first5=R. |last6=Mateczun |first6=A. |title=Specificity of an immunochromato-graphic test for anthrax |journal=Australian Veterinary Journal |date=April 2004 |volume=82 |issue=4 |pages=220–222 |doi=10.1111/j.1751-0813.2004.tb12682.x|pmid=15149073 }}</ref>

===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 [[Elution|elute]] the protein of interest include changing the pH, or adding a competitive molecule, such as [[imidazole]].<ref>{{cite book |last1=Singh|first1=Naveen K. |last2=DSouza|first2=Roy N. |last3=Bibi|first3=Noor S. |last4= Fernández-Lahore|first4=Marcelo |chapter=Direct Capture of His6-Tagged Proteins Using Megaporous Cryogels Developed for Metal-Ion Affinity Chromatography |year=2015 |editor1-last=Reichelt|editor1-first=S. |title=Affinity Chromatography |journal=<!-- Citation bot bypass--> |chapter-url=https://www.springer.com/us/book/9781493924462 |series=Methods in Molecular Biology |language=en |volume=1286 |location=New York |publisher=Humana Press |pages=201–212 |doi=10.1007/978-1-4939-2447-9_16 |pmid=25749956 |isbn=978-1-4939-2447-9}}</ref><ref>{{cite journal |last1= Gaberc-Porekar |first1= Vladka K.|last2= Menart |first2= Viktor|date=2001|title= Perspectives of immobilized-metal affinity chromatography|journal=J Biochem Biophys Methods |volume=49 |issue= 1–3 |pages= 335–360|doi= 10.1016/S0165-022X(01)00207-X|pmid= 11694288}}</ref>
[[File:Nickel resin.jpg|thumb|150px|A chromatography column containing nickel-agarose beads used for purification of proteins with histidine tags]]
{{See also|Polyhistidine-tag}}

===Recombinant proteins===
Possibly the most common use of affinity chromatography is for the purification of recombinant proteins. Proteins with a known affinity are [[protein tag]]ged 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 tag]]s include hexahistidine ([[histidine|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.<ref>{{Cite journal |last1=Vassylyeva |first1=Marina N. |last2=Klyuyev |first2=Sergiy |last3=Vassylyev |first3=Alexey D. |last4=Wesson |first4=Hunter |last5=Zhang |first5=Zhuo |last6=Renfrow |first6=Matthew B. |last7=Wang |first7=Hengbin |last8=Higgins |first8=N. Patrick |last9=Chow |first9=Louise T. |last10=Vassylyev |first10=Dmitry G. |date=2017-06-27 |title=Efficient, ultra-high-affinity chromatography in a one-step purification of complex proteins |journal=Proceedings of the National Academy of Sciences of the United States of America |volume=114 |issue=26 |pages=E5138–E5147 |doi=10.1073/pnas.1704872114 |doi-access=free |issn=0027-8424 |pmc=5495267 |pmid=28607052|bibcode=2017PNAS..114E5138V }}</ref>

===Lectins===
[[Lectin]] affinity chromatography is a form of affinity chromatography where [[lectin]]s 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.<ref name="Freeze Unit 9.1">{{Cite book|last=Freeze|first=H. H.|date=May 2001|chapter=Lectin affinity chromatography|title=Current Protocols in Protein Science |pages=9.1.1–9.1.9|doi=10.1002/0471140864.ps0901s00|issn=1934-3663|pmid=18429210|isbn=978-0471140863|s2cid=3197260}}</ref> Another example of a lectin is wheat germ agglutinin which binds D-N-acetyl-glucosamine.<ref name="Hage 1999">{{Cite journal|last=Hage|first=David|date=May 1999|title=Affinity Chromatography: A Review of Clinical Applications|url=http://clinchem.aaccjnls.org/content/clinchem/45/5/593.full.pdf|journal=Clinical Chemistry|volume=45|issue=5|pages=593–615|pmid=10222345|doi=10.1093/clinchem/45.5.593|doi-access=free}}</ref> The most common application is to separate [[glycoprotein]]s from non-glycosylated proteins, or one [[glycoform]] from another glycoform.<ref>{{cite web|url=http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/protein_purification~affinity~immobilized_lectin|title=GE Healthcare Life Sciences, Immobilized lectin|access-date=2010-11-29|archive-url=https://web.archive.org/web/20120303220910/http://www.gelifesciences.com/aptrix/upp01077.nsf/Content/protein_purification~affinity~immobilized_lectin|archive-date=2012-03-03|url-status=dead}}</ref> Although there are various ways to perform lectin affinity chromatography, the goal is extract a sugar ligand of the desired protein.<ref name="Freeze Unit 9.1"/>

=== 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.<ref>{{cite book|title=Fundamental Laboratory Approaches for Biochemistry and Biotechnology|last1=Ninfa|first1=Alexander J.|last2=Ballou|first2=David P.|last3=Benore|first3=Marilee|year=2006|edition=2nd|publisher=Wiley|page=153}}</ref>

==== 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.<ref>{{Cite book|title=Fundamental laboratory approaches for biochemistry and biotechnology|last1=Ninfa|first1=Alexander J.|last2=Ballou|first2=David P.|last3=Benore|first3=Marilee|date=2010|publisher=John Wiley|isbn=9780470087664|edition=2nd|location=Hoboken, N.J.|page=240|oclc=420027217}}</ref>

==== Boronate affinity chromatography ====
Boronate affinity chromatography consists of using boronic acid or boronates to elute and quantify amounts of [[glycoprotein]]s. Clinical adaptations have applied this type of chromatography for use in determining long term assessment of diabetic patients through [[Glycated hemoglobin#Measurement|analysis of their glycated hemoglobin]].<ref name="Hage 1999" />

=== Serum albumin purification ===
Affinity purification of albumin and macroglobulin contamination is helpful in removing excess albumin and α<sub>2</sub>-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<sup>−</sup>).<ref>{{Cite journal|last1=Naval|first1=Javier|last2=Calvo|first2=Miguel|last3=Lampreave|first3=Fermin|last4=Piñeiro|first4=Andrés|date=1983-01-01|title=Affinity chromatography of serum albumin: An illustrative laboratory experiment on biomolecular interactions|journal=Biochemical Education|language=en|volume=11|issue=1|pages=5–8|doi=10.1016/0307-4412(83)90004-3|issn=1879-1468}}</ref>