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Nebexpress ni spin columns

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The NEBExpress Ni Spin Columns are affinity chromatography columns designed for the purification of recombinant His-tagged proteins. The columns contain Ni-NTA agarose resin that binds to the histidine tag on the target protein, allowing for its isolation from the sample.

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Lab products found in correlation

Endo hf, by New England Biolabs (3 mentions) His tag antibody, by Thermo Fisher Scientific (3 mentions) Sonicated, by Qsonica (3 mentions) Vivaspin 30 kda mwco filters, by Sartorius (3 mentions) Glycobuffer 3, by New England Biolabs (3 mentions) Tnt quick coupled transcription translation system, by Promega (1 mentions) Adp glo kinase assay, by Promega (1 mentions)

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Ni spin columns (2 citations)

4 protocols using nebexpress ni spin columns

1

Purification and Glycosylation Analysis of Recombinant Proteins

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Example 4

The spent culture media (P. pastoris constructs) were buffer-exchanged in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and concentrated ten-fold using Vivaspin 30 kDa MWCO filters (Sartorius). To assess the extent of glycosylation, the concentrated spent culture media were subject to Endo Hf (New England Biolabs) digestions under native conditions in the presence of 1× GlycoBuffer 3 (50 mM sodium acetate, pH 6.0).

FCE proteins (wild-type and N-glycan variants) were purified from the concentrated spent cultures using NEBExpress Ni Spin Columns (New England Biolabs). The columns were washed twice with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 5 mM imidazole then once with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 10 mM imidazole. The purified protein was eluted with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 500 mM imidazole.

To prepare cell lysates (K. lactis constructs), cells were resuspended in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and sonicated (Qsonica). The soluble cell lysate was pre-cleared by centrifugation at 16000×g for 15 minutes at 4° C. The cell lysates and spent culture media were analyzed by SDS-PAGE on 4-20% polyacrylamide gel, followed by western blotting with a His-tag antibody (Thermo Fisher Scientific).

US11098295B1. FCE mRNA capping enzyme compositions, methods and kits (2021-08-24). New England Biolabs, Inc. [US]. Inventors: Mehul Ganatra [US], Siu-Hong Chan [US], Christopher H. Taron [US], G. B. Robb [US].
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2

Purification and Analysis of P. pastoris and K. lactis Proteins

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Example 4

The spent culture media (P. pastoris constructs) were buffer-exchanged in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and concentrated ten-fold using Vivaspin 30 kDa MWCO filters (Sartorius). To assess the extent of glycosylation, the concentrated spent culture media were subject to Endo Hf (New England Biolabs) digestions under native conditions in the presence of 1× GlycoBuffer 3 (50 mM sodium acetate, pH 6.0).

FCE proteins (wild-type and N-glycan variants) were purified from the concentrated spent cultures using NEBExpress Ni Spin Columns (New England Biolabs). The columns were washed twice with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 5 mM imidazole then once with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 10 mM imidazole. The purified protein was eluted with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 500 mM imidazole.

To prepare cell lysates (K. lactis constructs), cells were resuspended in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and sonicated (Qsonica). The soluble cell lysate was pre-cleared by centrifugation at 16000×g for 15 minutes at 4° C. The cell lysates and spent culture media were analyzed by SDS-PAGE on 4-20% polyacrylamide gel, followed by western blotting with a His-tag antibody (Thermo Fisher Scientific).

US11028379B1. FCE mRNA capping enzyme compositions, methods and kits (2021-06-08). New England Biolabs, Inc. [US]. Inventors: Mehul Ganatra [US], Siu-hong Chan [US], Christopher H. Taron [US], G. B. Robb [US].
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3

Purification and Analysis of P. pastoris and K. lactis Constructs

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Example 4

The spent culture media (P. pastoris constructs) were buffer-exchanged in 50 mM Tris-HC1, pH 7.5 buffer containing 300 mM NaCl and concentrated ten-fold using Vivaspin 30 kDa MWCO filters (Sartorius). To assess the extent of glycosylation, the concentrated spent culture media were subject to Endo Hf (New England Biolabs) digestions under native conditions in the presence of 1× GlycoBuffer 3 (50 mM sodium acetate, pH 6.0).

FCE proteins (wild-type and N-glycan variants) were purified from the concentrated spent cultures using NEBExpress Ni Spin Columns (New England Biolabs). The columns were washed twice with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 5 mM imidazole then once with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 10 mM imidazole. The purified protein was eluted with 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and 500 mM imidazole.

To prepare cell lysates (K. lactis constructs), cells were resuspended in 50 mM Tris-HCl, pH 7.5 buffer containing 300 mM NaCl and sonicated (Qsonica). The soluble cell lysate was pre-cleared by centrifugation at 16000×g for 15 minutes at 4° C. The cell lysates and spent culture media were analyzed by SDS-PAGE on 4-20% polyacrylamide gel, followed by western blotting with a His-tag antibody (Thermo Fisher Scientific).

US11725196B2. FCE mRNA capping enzyme compositions, methods and kits (2023-08-15). New England Biolabs, Inc. [US]. Inventors: Mehul Ganatra [US], Siu-hong Chan [US], Christopher H. Taron [US], G. B. Robb [US].
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4

Screening of GSK-3 Candidate Targets

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GSK-3 candidate targets are synthesized by TnT Quick coupled transcription/translation systems (Promega, # L1170). Two rounds of PCR reactions were used to generate cDNA templates with T7 promoter added in 5′ end and His-tag added in 3′ end. For the 1st round PCR reaction, forward primers with a part of T7 promoter sequence and reverse primers with His-tag sequence were used to generate intermediate PCR products. In the 2nd round PCR reaction, a common forward primer with the complete T7 promoter sequence and reverse primers were used to generate the final cDNA templates containing T7 promoter in 5′ end and His-tag in 3′ end for GSK3 candidate targets. After gel ∼500 ng PCR template is added to an aliquot of the TnT Quick Master Mix and incubated in a 50 µl reaction volume for 90 min at 30 °C. The synthesized proteins are purified by NEBExpress® Ni Spin Columns (NEB, #1427 S) and then analyzed by SDS-PAGE. ~20 ng synthesized candidate proteins were mixed with ∼40 ng recombinant GSK-3 protein (Sino Biological #10044-H07B) in a total 25 µl reaction volume. The in vitro phosphorylation reaction was performed following ADP-Glo™ Kinase Assay (Promega, # V9101). (primers sequences located in Supplementary Data 5)
Yue S., Wang L., DeMartino G.N., Zhao F., Liu Y, & Sieber M.H. (2022). Highly conserved shifts in ubiquitin-proteasome system (UPS) activity drive mitochondrial remodeling during quiescence. Nature Communications, 13, 4462.
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