Amylose resin

Manufactured by New England Biolabs

Sourced in United States, United Kingdom, Germany, Sweden, China, France

Amylose resin is a chromatography resin used for the purification of proteins and enzymes. It functions by selectively binding to proteins with a high affinity for amylose, a component of starch. This resin can be used to isolate and concentrate target proteins from complex mixtures.

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Lab products found in correlation

Ni nta agarose, by Qiagen (22 mentions) Glutathione sepharose 4b, by GE Healthcare (21 mentions) Pmal c5x vector, by New England Biolabs (12 mentions) Glutathione sepharose, by GE Healthcare (12 mentions) Pmal c2x, by New England Biolabs (10 mentions) Pmal c5x, by New England Biolabs (9 mentions) Glutathione beads, by GE Healthcare (9 mentions) Anti mbp, by New England Biolabs (9 mentions) Lightshift chemiluminescent emsa kit, by Thermo Fisher Scientific (9 mentions) Protease inhibitor cocktail, by Roche (9 mentions) Maltose, by Merck Group (8 mentions) Pmal c2x vector, by New England Biolabs (8 mentions) Glutathione sepharose 4b beads, by GE Healthcare (8 mentions) Ni nta resin, by Qiagen (7 mentions) Anti mbp antibody, by New England Biolabs (6 mentions) Superdex 200, by GE Healthcare (6 mentions) Glutathione sepharose bead, by GE Healthcare (6 mentions) Glutathione resin, by GE Healthcare (6 mentions) Glutathione resin, by GenScript (6 mentions) Turbofect, by Thermo Fisher Scientific (6 mentions)

527 protocols using amylose resin

Purification of MBP-tagged MVV VCBC Complexes

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Small-scale expressions of unfused MBP-tagged MVV VCBC wild type (WT) and variants were induced in 50 ml of Terrific Broth by 0.3 mM IPTG at 16°C for 16 hours. Cells were harvested and lysed by sonication. The lysate was clarified by centrifugation and then purified by 50 μl of Ni-NTA (Qiagen) beads in a 1.5-ml Eppendorf tube. The Ni-NTA eluate was further mixed with 50 μl of amylose resin (New England BioLabs) and incubated for 1 hour. After removing the supernatant by centrifugation, the resin was washed with 300 μl of binding buffer [30 mM tris, 100 mM NaCl, and 0.2 mM TCEP (pH 8.0)] for three times. Eighty microliters of the elution buffer (binding buffer plus 0.2 mM maltose) was then added to the resin and incubated at 4°C for 10 min before centrifugation. The loading and elution fractions were analyzed by SDS-PAGE.
A total of 0.15 mg of MBP-tagged unfused MVV VCBC or fused Bril-tagged VCBC complexes (0.15 mg) was first incubated with human mCherry-A3H at 1:2 molar ratio in 100 μl of binding buffer containing 30 mM tris, 100 mM NaCl, and 0.2 mM TCEP (pH 8.0) at 4°C for 2 hours, subsequently mixed with 50 μl of amylose resin (New England BioLabs) in 1.5-ml EP tubes, and incubated for one additional hour. The subsequent steps for A3H/VCBC complex purification were the same as above. The loading and elution fractions were analyzed by SDS-PAGE.

Hu Y., Gudnadóttir R.B., Knecht K.M., Arizaga F., Jónsson S.R, & Xiong Y. (2023). Structural basis for recruitment of host CypA and E3 ubiquitin ligase by maedi-visna virus Vif. Science Advances, 9(2), eadd3422.

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Characterization of hFip1-CPSF30 Interactions

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For pull-down analysis of hFip1 mutants binding to CPSF30, 120 µg of purified His₆-MBP-TEV-CPSF30^1–243 wt protein was incubated with 120 µl amylose resin (NEB) equilibrated in pull-down wash buffer and gently agitated at 4°C for 1 hr. The beads were washed three times with 0.5 ml of pull-down wash buffer and equally distributed in four tubes. His₆-GFP-TEV-hFip1^1–195 wt and point mutants (W150E, F161E, and W170E) were added in fivefold molar excess to the beads. After incubation at 4°C for 1 hr, gently agitated, unbound protein was washed off by adding three times 0.5 ml pull-down wash buffer. For pull-down analysis of the hFip1 interaction with the ZF of CPSF30, 15 µg of His₆-MBP-TEV-CPSF30^1–243 wt and ZF mutants were incubated each with 30 µl amylose resin (NEB) equilibrated in pull-down wash buffer and gently agitated at 4°C for 1 hr. Unbound protein was washed off three times with 0.5 ml of pull-down wash buffer and His₆-GFP-TEV-hFip1^1–195 wt was added in fourfold molar excess to the resin and incubated at 4°C for 1 hr, gently agitated. Beads were washed three times with 0.5 ml pull-down wash buffer.

Muckenfuss L.M., Migenda Herranz A.C., Boneberg F.M., Clerici M, & Jinek M. (2022). Fip1 is a multivalent interaction scaffold for processing factors in human mRNA 3′ end biogenesis. eLife, 11, e80332.

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Purification and Binding Analysis of Nup Complex

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The MBP-Nup62(322–525)·Nup54(346–510)·Nup58(239–415) complex from R. norvegicus was generated by co-expression of the proteins using a polycistronic vector where N termini of Nup58 and Nup62 were tagged to His₆ and maltose-binding protein (MBP) tags, respectively. The MBP-Nup62·Nup54·Nup58 complex was purified through His₆ affinity chromatography. The His₆ tag of Nup58 was cleaved with thrombin. The sample was dialyzed in buffer A. The purified protein complex was bound to amylose resin (New England Biolabs Inc.) equilibrated with buffer A. The MBP-Nup62·Nup54·Nup58-amylose resin was washed with buffer A and used for subsequent studies. 75 μl of amylose-MBP-Nup62·Nup54·Nup58 resin was incubated in a 0.8-ml centrifuge column (Thermo Scientific), at 4 °C with increasing amounts of wild-type Nup54 (453–494) in a final volume of 375 μl. The mixture was then spun at 1000 × g for 2 min at 4 °C. The amount of Nup58 released from the amylose matrix into the supernatant, as a function of the concentration of wild-type Nup54 (453–494), was calculated through absorbance of the supernatant at 280 nm, after the blank subtraction.

Sharma A., Solmaz S.R., Blobel G, & Melčák I. (2015). Ordered Regions of Channel Nucleoporins Nup62, Nup54, and Nup58 Form Dynamic Complexes in Solution. The Journal of Biological Chemistry, 290(30), 18370-18378.

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Affinity Purification of CRISPR Components

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100 μg MBP-SpyCas9 and 100 μg AcrIIA2/AcrIIA4, in presence or absence of sgRNA (molar ratio, 1:1.1), and 30 μl amylose resin (New England Biolabs) were mixed at 4°C for 1 hr in buffer D (20 mM HEPES, pH 7.2, 200 mM NaCl, 2 mM MgCh, 5 mM DTT). Alternately, 100 μg MBP-AcrIIA4 and 100 μg SpyCas9, in presence or absence of sgRNA (molar ratio, 1:1.1) and sgRNA-dsDNA (molar ratio, 1:1.1:1.5), and 30 μl amylose resin (NEB) were mixed at 4°C for 1 hr in buffer D. The resin was washed three times using buffer D and detected by SDS-PAGE.

Yang H, & Patel D.J. (2017). Inhibition Mechanism of an Anti-CRISPR Suppressor AcrIIA4 Targeting SpyCas9. Molecular cell, 67(1), 117-127.e5.

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Purification and Analysis of MBP-DmAMP1W Fusion Protein

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The pMAL-C5X-DmAMP1W and pMAL-C5X were transformed in E. coli DE3 competent cells. The transformed competent cells were grown in 200 mL LB medium (100 μg/mL ampicillin) until OD600 was 0.6, and then transformed competent cells were induced with 0.1 mM concentrations of IPTG under 28 °C for 12 h at 180 rpm.
The culture was divided into 50 mL tubes and then centrifuged at 4000 × g for 20 min to harvest cells. The cells were suspended in PBS buffer (pH 7.4) and divided in 1.5 mL tubes with 10 μL lysozyme (Thermo Fisher Scientific, Boston, MA, USA). Then, the cells were broken up by freeze-thaw method. The cell debris was centrifuged for 15 min at 4 °C and 12,000 × rpm. The clear supernatant, containing soluble fraction, was collected and purified; 100 μL Amylose Resin (NEB, Ipswich, MA, USA) was flowed with PBS buffer (pH 7.4) 3 times, and then the supernatant solution was incubated with Amylose Resin at 4 °C with end-over-end rotation overnight. The MBP and MBP-DmAMP1W were eluted by the MBP elution buffer (0.04% maltose solution), respectively. BCA method was used to confirm the protein concentration referring to the Easy II Protein Quantitative Kit (TransGen Biotech, Beijing, China) instruction. After that, a total of 10 μL of the purified proteins were analyzed by 12% SDS–PAGE (Bio-Rad, Hercules, CA, USA).

Su Q., Wang K, & Zhang Z. (2020). Ecotopic Expression of the Antimicrobial Peptide DmAMP1W Improves Resistance of Transgenic Wheat to Two Diseases: Sharp Eyespot and Common Root Rot. International Journal of Molecular Sciences, 21(2), 647.

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Cargo-AP-1 Interaction Assay

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Interaction of cargo proteins with the AP-1 core in the absence or presence of Arf1∆1-16-Q71L was analyzed using a previously described pull-down assay (Guo et al., 2013 (link); Ren et al., 2013 (link)). In brief, MBP-tagged cargo protein (20 μg) immobilized on amylose resin (New England Biolabs, Beverly, MA) was incubated with 0.05 μM AP-1 core precleared on amylose resin in the absence or presence of 10 μM Arf1∆1-16-Q71L and 1 mM GTP. The pull down was analyzed by SDS–PAGE, followed by immunoblotting.

Jain S., Farías G.G, & Bonifacino J.S. (2015). Polarized sorting of the copper transporter ATP7B in neurons mediated by recognition of a dileucine signal by AP-1. Molecular Biology of the Cell, 26(2), 218-228.

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Affinity Purification of LcMYB4 Interactome

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To obtain the protein interacting with LcMYB4, the ORF sequence of LcMYB4 was cloned into the pET-30a expression vector (Novagen, Madison, WI) to produce His₆ tag-fused LcMYB4. The LcMYB4-His₆ fusion protein was coupled to amylose resin (New England Biolabs) according to the manufacturer’s instructions, and then the protein extracts of sheepgrass seedlings were incubated with LcMYB4-His₆–amylose resin at 4 °C for 4 h. Subsequently, the nonspecific binding proteins were washed five times with imidazole buffer (10 mM–100 mM) with different concentration gradients. The bound proteins were eluted with SDS/PAGE sample buffer and resolved by SDS/PAGE followed by mass spectrometry [69 (link), 70 ].

Li X., Jia J., Zhao P., Guo X., Chen S., Qi D., Cheng L, & Liu G. (2020). LcMYB4, an unknown function transcription factor gene from sheepgrass, as a positive regulator of chilling and freezing tolerance in transgenic Arabidopsis. BMC Plant Biology, 20, 238.

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Affinity Purification and Characterization of AtNBR1-KEAP1 Interactions

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MBP-tagged AtNBR1–PB1^1–94 and mutant AtNBR1 residues 1–94 (AtNBR1–PB1) were expressed as described above and buffer-exchanged into 15 mM Tris (pH 7.5), 150 mM NaCl. For the AtNBR1 pull-down experiments, 50 μl of amylose resin (NEB) was incubated for 10 min with MBP–AtNBR1–PB1, followed by 5-min incubations with a 4:1 molar excess of mutant AtNBR1–PB1. Beads were washed with 15 mM HEPES (pH 7.5), 500 mM NaCl, eluted with 15 mM HEPES (pH 7.5), 150 mM NaCl, and 30 mM maltose, and fractions were analyzed by SDS-PAGE. p62, TFG1-mini-p62, and TFG1-p62 were purified as described and applied to size-exclusion chromatography on a Superdex 75 16/60 in 20 mM Tris (pH 8), 100 mM NaCl. Human KEAP1–DC^309–624 was cloned into pET-28a(+) with a N-terminal 6×His tag, purified on Ni-NTA resin, and buffer-exchanged into 20 mM Tris (pH 8), 100 mM NaCl. Approximately 100 μl of amylose resin (NEB) was incubated for 30 min with either of the MBP-containing proteins at room temperature, followed by 30-min incubations with a 3:1 molar excess of KEAP1–DC^309–624. Beads were washed with 20 mM Tris (pH 8), 1 M NaCl, and eluted with 20 mM Tris (pH 8), 100 mM NaCl, and 20 mM maltose. Fractions were analyzed by SDS-PAGE.

Jakobi A.J., Huber S.T., Mortensen S.A., Schultz S.W., Palara A., Kuhm T., Shrestha B.K., Lamark T., Hagen W.J., Wilmanns M., Johansen T., Brech A, & Sachse C. (2020). Structural basis of p62/SQSTM1 helical filaments and their role in cellular cargo uptake. Nature Communications, 11, 440.

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In vitro Phosphorylation Assays of Barley Proteins

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In vitro phosphorylation assays were performed as described previously (Ma et al., 2021) . The pET-N, pET-N S290A , and pET-N S290D plasmids were transformed into E. coli (BL21) and N-His, N S290A -His, N S290D -His proteins were purified by Ni-NTA Agarose (QIAGEN). Total soluble proteins were extracted from young leaves of healthy barley plants (HvEX) and used as kinase resource, and then 1-μg HvEX was mixed with 10-μg N-His/N S290A -His/N S290D -His proteins and incubated at 30 C for 40 min. MBP-HvMPK3, MBP-HvMPK3 m , or MBP-LsERK were purified using amylose resin (NEB) and incubated with GST-HvMPKK in reaction (50-mM Tris-HCl, pH 7.5, 10-mM MgCl 2 , 1-mM DTT, and 0.2-mM ATP) for 30 min at 30 C. Then GST-HvMPKK was removed using glutathione-agarose beads (GE Healthcare) and the pre-activated HvMPK3, MBP-HvMPK3 m , or MBP-LsERK were purified using amylose resin (NEB). For in vitro phosphorylation assays, pre-activated MBP-HvMPK3, MBP-HvMPK3 m , MBP-LsERK, or MBP were incubated with N-His, N S290A -His, N S290D -His in 30 μL 1 kinase reaction buffer (25-mM Tris-HCl, pH 7.5, 10-mM MgCl 2 , 10-mM CaCl 2 , 10-mM DTT, 0.025-mM ATP, and 5 μCi of 32 P-γ-ATP) at 30 C for 30 min. Next, 5 μL 5 SDS sample buffer was added to terminate the reactions. The proteins were separated on a 12.5% SDS-PAGE gel. The 32 P-γ-ATP-labeled phosphorylated proteins were visualized by autoradiography.

Ding Z.H., Gao Q., Tong X., Xu W.Y., Ma L., Zhang Z.J., Wang Y, & Wang X.B. (2022). MAPKs trigger antiviral immunity by directly phosphorylating a rhabdovirus nucleoprotein in plants and insect vectors. The Plant cell, 34(8).

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Purification of MBP-TLR9 TIR Domain and Beclin 1

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The maltose-binding domain (MBP)-tagged TLR9 TIR domain (amino acids 868–1032) plasmid (gift of J. Hurley, UC Berkeley) was used to re-clone the MBP-TLR9 TIR domain into the pCAG vector, removing the twin-Strep and Flag tags. One liter of HEK 293S cells (3.0 × 10⁶ cells/ml) was transfected with the MBP-TIR plasmid using polyethylenimine (PEIpro) in the presence of 10 mM sodium butyrate (Sigma-Aldrich). After shaking for 48 h at 37°C and 8% CO₂, cells were harvested and lysed by adding 1% Triton X-100, followed by centrifugation at 50,000 x g for 30 min. The clear supernatant was collected, amylose resins (NEB) were added and incubated for 1 h at 4°C. The resins were washed with 15 column volumes of the purification buffer (20 mM Tris pH 8.0, 150 mM NaCl, 1 mM DTT). The protein was eluted with purification buffer containing 10 mM maltose (Sigma-Aldrich) and subjected to size exclusion chromatography. The eluate was pooled, concentrated, flash-frozen by liquid nitrogen and stored at −80°C. The bacterial expression plasmid containing full-length human BECN 1 (gift from Dr. Matthew J. Ranaghan, the Broad Institute of Harvard and MIT) was used to express and purify Beclin 1 protein as described^{25 (link)}.

Liu Y., Nguyen P.T., Wang X., Zhao Y., Meacham C.E., Zou Z., Bordieanu B., Johanns M., Vertommen D., Wijshake T., May H., Xiao G., Shoji-Kawata S., Rider M.H., Morrison S.J., Mishra P, & Levine B. (2020). Tlr9 and Beclin 1 Cross-Talk Regulates Muscle AMPK Activation in Exercise. Nature, 578(7796), 605-609.

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