Abstract
Cellular membrane affinity chromatography is a technique that is based on the immobilization of a target trans-membrane protein onto a stationary phase. The target protein is isolated by homogenization and solubilization of a source (e.g., cell line) followed by immobilization on either the immobilized artificial membrane-phosphatidyl choline (IAM-PC) stationary phase or the surface of an open tubular capillary during a dialysis step. The procedure typically takes 3–4 d for the IAM-PC stationary phase, whereas the open-tubular method takes an extra week for the preparation of the capillary. The resulting columns can then be used to characterize binding sites on the target protein through frontal chromatographic and/or nonlinear chromatographic studies using a wide variety of ligands including small molecules and polypeptides. The columns have been used in drug discovery as well as in the screening of tobacco smoke condensates.
This is a preview of subscription content, access via your institution
Access options
Subscribe to this journal
Receive 12 print issues and online access
$259.00 per year
only $21.58 per issue
Buy this article
- Purchase on Springer Link
- Instant access to full article PDF
Prices may be subject to local taxes which are calculated during checkout
Similar content being viewed by others
References
Ng, E.S.M., Chora, N.W.C., Lewis, D.F., Hindsgaul, O. & Schriemer, D. Frontal affinity chromatography-mass spectrometry. Nat. Protoc. 2, 1907–1917 (2007).
Chaiken, I.M. Analytical affinity chromatography in studies of molecular recognition in biology: a review. J. Chromatogr. 376, 11–32 (1986).
Wade, J.L., Bergold, A.F. & Carr, P.W. Theoretical description of nonlinear chromatography, with applications to physicochemical measurements in affinity chromatography and implications for preparative-scale separations. Anal. Chem. 59, 1286–1295 (1987).
Kim, H.S., Kye, Y.S. & Hage, D.S. Development and evaluation of N-hydroxysuccinimide-activated silica for immobilizing human serum albumin in liquid chromatography columns. J. Chromatogr. A. 1049, 51–61 (2004).
Moaddel, R. et al. Automated ligand fishing using HSA-coated magnetic beads. Anal. Chem. 79, 5414–5417 (2007).
Brekkan, E., Lundqvist, A. & Lundahl, P. Immobilized membrane vesicle or proteoliposome affinity chromatography. Frontal analysis of interactions of cytochalasin B and D-glucose with the human red cell glucose transporter. Biochemistry 35, 12141–12145 (1996).
Yang, Q. & Lundahl, P. Immobilized proteoliposome affinity chromatography for quantitative analysis of specific interactions between solutes and membrane proteins. interaction of cytochalasin B and D-glucose with the glucose transporter Glut1. Biochemistry 34, 7289–7294 (1995).
Zhang, Y., Xiao, Y.X., Kellar, K.J. & Wainer, I.W. Immobilized nicotinic receptor stationary phase for on-line liquid chromatographic determination of drug-receptor affinities. Anal. Biochem. 264, 22–25 (1998).
Pidgeon, C., Marcus, C. & Alvarez, F. Applications of Enzyme Biotechnology, (eds. Baldwin, T.O. & Kelly, J.W.) (Plenum Press, New York, 1992).
Besanger, T.R., Hodgson, R.J., Green, J.R.A. & Brennan, J.D. Immobilized enzyme reactor chromatography: optimization of protein retention and enzyme activity in monolithic silica stationary phases. Analytica. Chimica. Acta. 564, 106–115 (2006).
Beigi, F. & Wainer, I.W. Syntheses of immobilized G protein-coupled receptor chromatographic stationary phases: characterization of immobilized mu and kappa opioid receptors. Anal. Chem. 75, 4480–4485 (2003).
Beigi, F., Chakir, K., Xiao, R.-P. & Wainer, I.W. G-protein-coupled receptor chromatographic stationary phases. 2. Ligand-induced conformational mobility in an immobilized beta2-adrenergic receptor. Anal. Chem. 76, 7187–7193 (2004).
Moaddel, R., Calleri, E., Massolini, G., Frazier, C. & Wainer, I.W. The synthesis and initial characterization of an immobilized purinergic receptor (P2Y1) liquid chromatography stationary phase for on line screening. Anal. Biochem. 364, 216–218 (2007).
Moaddel, R., Jozwiak, K., Whittington, K.C. & Wainer, I.W. The on-line determination of agonists/ competitive antagonists and non-competitive inhibitors of the α3β2, α3β4, α4β2, and α4β4 -nicotinic acetylcholine receptors using immobilized receptor-based liquid chromatographic stationary phases. Anal. Chem. 77, 895–901 (2005).
Zhang, Y., Leonessa, F., Clarke, R. & Wainer, I.W. Development of an immobilized P-glycoprotein stationary phase for on-line liquid chromatographic determination of drug-binding affinities. J. Chromatogr. B. 739, 33–37 (2000).
Moaddel, R., Yamaguchi, R., Ho, P., Patel, S. & Wainer, I.W. Development and characterization of the human organic cation transporter column. J. Chrom. B. 818, 263–268 (2005).
Kimura, T., Perry, J., Anzai, N., Pritchard, J. & Moaddel, R. Development and characterization of immobilized human organic anion transporter based liquid chromatographic stationary phase: hOAT1 and hOAT2. J. Chrom. B. 859, 267–271 (2007).
Moaddel, R., Cloix, J.F., Ertem, G. & Wainer, I.W. Multiple receptor liquid chromatographic stationary phases: development of a co-immobilized nicotinic receptor, GABA receptor and NMDA receptor stationary phase. Pharm. Res. 19, 104–107 (2002).
Moaddel, R., Bullock, P. & Wainer, I.W. Development and characterization of an open tubular column containing immobilized P-glycoprotein for rapid on-line screening for P-glycoprotein substrates. J. Chrom. B. Analyt. Technol. Biomed. Life Sci. 799, 255–263 (2004).
Maciuk, A., Moaddel, R., Haginaka, J. & Wainer, I.W. Screening of tobacco smoke condensate for nicotinic acetylcholine receptor ligands using cellular membrane affinity chromatography columns and missing peak chromatography. J. Pharm. Biomed. Anal. 48, 238–246 (2008).
Moaddel, R. et al. On-line screening of conformationally constrained nicotines and anabasines for agonist activity at the α3β4 and α4β2 -nicotinic acetylcholine receptors using immobilized receptor based liquid chromatographic stationary phases. J. Chrom. B. 813, 235–240 (2004).
Moaddel, R., Jozwiak, K. & Wainer, I.W. Allosteric modifiers of neuronal nicotinic acetylcholine receptors: new methods, new oppurtunities. Med. Res. Rev. 27, 723–753 (2007).
Moaddel, R., Hamid, R., Patel, S., Wainer, I.W. & Bullock, P. Comparison of an open tubular column containing immobilized P-glycoprotein with Caco-2 cell monolayers for the purpose of investigating interactions between drug candidates and Pgp. Anal. Chimica. Acta. 578, 25–30 (2006).
Moaddel, R., Ravichandran, S., Bighi, F., Yamaguchi, R. & Wainer, I.W. The Prediction and description of stereoselective binding to the human organic cation transporter (hOCT1). Brit. J. Pharm. 151, 1305–1314 (2007).
Jozwiak, K., Hernandez, S.C., Kellar, K.J. & Wainer, I.W. Enantioselective interactions of dextromethorphan and levomethorphan with the α3β4-nicotinic acetylcholine receptor: comparison of chromatographic and functional data. J. Chromatogr. B. 797, 373–379 (2003).
Jozwiak, K., Ravichandran, S., Collins, J., Moaddel, R. & Wainer, I.W. Interaction of noncompetitive inhibitors of the α3β2 nicotinic acetylcholine receptor investigated by affinity chromatography and molecular docking. J. Med. Chem. 50, 6279–6283 (2007).
Kitabatake, T. et al. Characterization of a multiple ligand-gated ion channel cellular membrane affinity chromatography (CMAC) column and identification of endogenously expressed receptors in astrocytoma cell lines. Anal. Chem. 80, 8673–8680 (2008).
Moaddel, R. et al. Initial synthesis and characterization of an a7 nicotinic receptor cellular membrane affinity chromatography column: effect of receptor subtype and cell type. Anal. Chem. 80, 48–54 (2008).
Leonessa, F. et al. Effect of tamoxifen on the multidrug-resistant phenotype in human breast cancer cells: isobologram, drug accumulation, and M(r) 170,000 glycoprotein (gp170) binding studies. Cancer Res. 54, 441–447 (1994).
Walsch, C.T. Posttranslational Modifications of Proteins: Expanding Nature's Inventory (Roberts and Co., Greenwood, CO, USA, 2007).
Wade, L., Bergold, A.F. & Carr, P.W. Theoretical description of nonlinear chromatography, with applications to physicochemical measurements in affinity chromatography and implications for preparative-scale separations. Anal. Chem. 59, 1286–1295 (1986).
Baynham, M.T., Patel, S., Moaddel, R. & Wainer, I.W. Multidimensional on-line screening for ligands to the α3β4 neuronal nicotinic acetylcholine receptor using an immobilized nicotinic receptor liquid chromatographic stationary phase. J. Chrom. B. 772, 155–61 (2002).
Moaddel, R. et al. utomated ligand fishing using HSA-coated magnetic beads. Anal. Chem. 79, 5414–5417 (2007).
Marszall, M. et al. Ligand and protein fishing with heat shock protein 90 coated magnetic beads. Anal. Chem. 80, 7571–7575 (2008).
Cleland, J.L. et al. A specifc molar ratio of stabilizer to protein is required for storage stability of a lyophilized monoclonal antibody. J. Pharm. Sci. 90, 310–321 (2000).
Acknowledgements
This work was funded by the Intramural Research Program of the National Institute on Aging/NIH.
Author information
Authors and Affiliations
Corresponding author
Rights and permissions
About this article
Cite this article
Moaddel, R., Wainer, I. The preparation and development of cellular membrane affinity chromatography columns. Nat Protoc 4, 197–205 (2009). https://doi.org/10.1038/nprot.2008.225
Published:
Issue Date:
DOI: https://doi.org/10.1038/nprot.2008.225
This article is cited by
-
Preparation and characterization of micro-cell membrane chromatographic column with silica-based porous layer open tubular capillary as cellular membrane carrier
Analytical and Bioanalytical Chemistry (2016)
-
Preparation and Application of Modified VEGFR-2 Cell Membrane Chromatographic Separation System
Chromatographia (2016)
-
Stabilized phospholipid membranes in chromatography: toward membrane protein-functionalized stationary phases
Analytical and Bioanalytical Chemistry (2014)
-
Development of new chromatographic tools based on A2A adenosine receptor subtype for ligand characterization and screening by FAC-MS
Analytical and Bioanalytical Chemistry (2013)
-
Recent developments in protein–ligand affinity mass spectrometry
Analytical and Bioanalytical Chemistry (2011)
Comments
By submitting a comment you agree to abide by our Terms and Community Guidelines. If you find something abusive or that does not comply with our terms or guidelines please flag it as inappropriate.