Amylose affinity column

Manufactured by New England Biolabs

The Amylose Affinity Column is a chromatography tool used to purify proteins that contain an affinity tag derived from the carbohydrate amylose. The column consists of cross-linked agarose beads that have been covalently linked to amylose. Proteins with an amylose-binding tag will bind to the column, allowing for their separation and purification from other components in a sample.

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Lab products found in correlation

Q sepharose column, by GE Healthcare (1 mentions) Superdex 75 size exclusion column, by GE Healthcare (1 mentions) Hitrap q, by GE Healthcare (1 mentions) Hitrap s, by GE Healthcare (1 mentions) Hitrap, by Cytiva (1 mentions) Hiscribe t7 arca mrna kit, by New England Biolabs (1 mentions) T4 ligase, by New England Biolabs (1 mentions) Quickchange 2 xl site directed mutagenesis kit, by Agilent Technologies (1 mentions) Mmessage mmachine t7 ultra transcription kit, by Thermo Fisher Scientific (1 mentions) Pmalc2t vector, by New England Biolabs (1 mentions) Gibson assembly, by New England Biolabs (1 mentions)

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Amylose column (9 citations) Amylose affinity (2 citations)

4 protocols using amylose affinity column

Purification of Recombinant Calmodulin

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Full-length human and C. elegans calmodulin were expressed in E. coli Rosetta (DE3), both from a modified pET vector containing an N-terminal maltose-binding protein (MBP) and a tobacco etch virus (TEV) cleavage site. The MBP–TEV–calmodulin fusion protein was purified on an amylose affinity column (New England Biolabs) and subsequently cleaved by a TEV protease. Following cleavage, calmodulin, TEV and MBP were separated by anion ion exchange chromatography using a Q-sepharose column (GE Healthcare). The calmodulin-containing fractions were pooled and concentrated before removing residual Ca²⁺ by adding 20 mm ethylenediaminetetraacetic acid (EDTA) to the sample prior to applying it to a Superdex 75 size exclusion column (GE Healthcare). The identity and integrity of each protein preparation was confirmed by matrix-assisted laser desorption/ionization-time of flight mass spectrometry. Protein concentrations were determined by absorption at 280 nm.

Jensen H.H., Frantzen M.T., Wesseltoft J.L., Busuioc A.O., Møller K.V., Brohus M., Duun P.R., Nyegaard M., Overgaard M.T, & Olsen A. (2023). Human calmodulin mutations cause arrhythmia and affect neuronal function in C. elegans. Human Molecular Genetics, 32(12), 2068-2083.

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Purification of MBP-GLD-1 Fusion Proteins

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The plasmids encoding pHMTc-GLD(135–336) or mutations thereof were transformed into Escherichia coli strain BL21(DE3). All three maltose-binding protein-GLD-1 fusions were purified as previously described (Ryder et al., 2004 (link)). Briefly, bacterial cultures were induced at mid log phase for three hours using 1 mM isopropyl 1-thio-β-D-galactopyranoside (IPTG). After harvesting, the cell pellets were lysed, then soluble recombinant protein was purified across an amylose affinity column (New England Biolabs) followed by HiTrap Q and HiTrap S ion exchange columns (GE Healthcare, Wauwatosa, WI) at 4°C. Relative purity of the protein was assessed by SDS-PAGE, and the concentration estimated from the absorption of 280 nm UV light using extinction coefficients calculated from the sequence composition with Expasy Protparam (Wilkins et al., 1999 ). Purified proteins were dialyzed into storage buffer (50 mM Tris pH 8.0, 20 mM NaCl, 2 mM DTT) and kept at 4°C until use.

Hu S., Ryan L.E., Kaymak E., Freeberg L., Lo T.W., Kuersten S., Ryder S.P, & Haag E.S. (2018). Multi-modal regulation of C. elegans hermaphrodite spermatogenesis by the GLD-1-FOG-2 complex. Developmental biology, 446(2), 193-205.

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Synthesis and Purification of zELK Channel Constructs

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The full-length Danio rerio zELK construct (GI: 159570347) was synthesized (Bio Basic) and subcloned into a modified pcDNA3 vector that contained a C-terminal YFP, a T7 promoter, and 3′ and 5′ untranslated regions of a Xenopus laevis β-globin gene. Point mutations were made using the Quickchange II XL Site-Directed Mutagenesis kit (Agilent Technologies). The deletions were made using standard overlapping PCR followed by ligation using T4 ligase or Gibson Assembly (New England Biolabs). The sequences of the DNA constructs were confirmed by fluorescence-based DNA sequencing (Genewiz). The RNA was synthesized in vitro using the HiScribe T7 ARCA mRNA kit (New England Biolabs) or the mMESSAGE mMACHINE T7 ULTRA Transcription kit (ThermoFisher) from the linearized cDNA.
Purification of maltose-binding protein (MBP)–C-linker/CNBHD proteins was done as previously described (Brelidze et al., 2009 (link), 2012 (link)). The C-linker/CNBHD proteins from zELK channels (amino acids Q543–L750) with or without Y740–L742 were subcloned into a modified pMalc2T vector (New England Biolabs) containing an N-terminal MBP affinity tag. The proteins were expressed in BL21(DE3) Escherichia coli cells and purified on an amylose affinity column (New England Biolabs).

Dai G., James Z.M, & Zagotta W.N. (2018). Dynamic rearrangement of the intrinsic ligand regulates KCNH potassium channels. The Journal of General Physiology, 150(4), 625-635.

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Generation and Characterization of Monoclonal Antibodies

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Rat monoclonal antibody GCN-3D12 was directed against aa 86–197 in the N-terminal domain of the GC from A. punctulata. A fusion protein comprising an N-terminal maltose-binding protein and aa 86–197 of the GC was generated and heterologously expressed in Escherichia coli. After purification with an amylose affinity column (New England Biolabs, Inc.), rats were immunized subcutaneously and intraperitoneally with the fusion protein in the presence of Freund’s adjuvant. A boost without adjuvant was given 6 wk after the primary injection. Fusion was performed using standard procedures. Supernatants were tested by differential ELISA with the GC–maltose-binding protein fusion protein and with unrelated proteins coupled to the same carrier. Monoclonal antibodies that reacted specifically with GC were further analyzed by Western blotting. Tissue culture supernatant of clone 3D12, rat IgG1 subclass, was used in this study. The antibody recognized the recombinant and the native protein both in the phosphorylated and unphosphorylated states. Mouse monoclonal antibody CRO/CBL 3B10-121 directed against aa 482–664 of the rat CNGA2 channel was generated following the same procedure (Meyer et al., 2000 (link)).

Pichlo M., Bungert-Plümke S., Weyand I., Seifert R., Bönigk W., Strünker T., Kashikar N.D., Goodwin N., Müller A., Körschen H.G., Collienne U., Pelzer P., Van Q., Enderlein J., Klemm C., Krause E., Trötschel C., Poetsch A., Kremmer E, & Kaupp U.B. (2014). High density and ligand affinity confer ultrasensitive signal detection by a guanylyl cyclase chemoreceptor. The Journal of Cell Biology, 206(4), 541-557.

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