his tag antibody, by Thermo Fisher Scientific - Product details

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

Detecting Recombinant Protein Expression

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The His-tag antibody (Beyotime, Shanghai, China) was used to test whether the inserted gC was successfully expressed due to the gC gene linked with His-tag. BHK-21 cells were respectively inoculated with the parental strain, the triple-gene deletion virus, and the triple-gene deletion plus gC virus (MOI = 0.01) and collected at 48 hpi. Proteins were obtained from infected cells using radioimmunoprecipitation assay lysis buffer (Thermo Fisher, Waltham, MA, USA). The same amounts of proteins were separated by 10% SDS-PAGE and subsequently transferred onto PVDF membrane. The membrane was incubated with His-tag antibody, followed by incubation with HRP-labeled secondary antibody (Thermo Fisher, Massachusetts USA). NcmECL Ultra Luminol/Enhancer Reagent (A) and NcmECL Ultra Stabilized Peroxide Reagent (B) were used to display the bands (NCM Biotech, Suzhou, China) and observed by exposure instrument (Tanon, Shanghai, China).

Wu Z., Deng J., Chen M., Lu P., Yan Z., Wu X., Ji Q., Fan H., Luo Y, & Ju C. (2024). Additional Insertion of gC Gene Triggers Better Immune Efficacy of TK/gI/gE-Deleted Pseudorabies Virus in Mice. Viruses, 16(5), 706.

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4

Immunodetection of E. chaffeensis ECH_0379 Protein

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For the detection of recombinant ECH_0379 protein, the above-described SDS-PAGE resolved proteins were transferred onto a nylon membrane (Thermo Fisher Scientific, Waltham, MA, USA) by subjecting them to electro-blotting using an electrophoretic transfer unit (Bio-Rad, Hercules, CA, USA). Subsequently, the presence of recombinant protein was assessed using a His-tag antibody as per the manufacturer instructions (Thermo Fisher, Rockford, IL, USA). The RC and DC forms of E. chaffeensis were purified by subjecting them to renografin density gradient centrifugation, as described previously [44 (link)]. About 20 µg each of the total E. chaffeensis proteins from the RC and DC fractions were resolved on an SDS-PAGE and transferred to a nylon membrane, as indicated above, and used for blot analysis with the ECH_0379-specific polyclonal antibody. Polyclonal serum was raised in rabbits against ECH_0379 recombinant protein using a fee-for-service facility (Thermo Fisher, Waltham, MA, USA). A secondary anti-rabbit antibody conjugated with horseradish peroxidase (Sigma-Aldrich, St. Louis, MO, USA) was used for the signal detection.

Wei L., Liu H., Alizadeh K., Juarez-Rodriguez M.D, & Ganta R.R. (2021). Functional Characterization of Multiple Ehrlichia chaffeensis Sodium (Cation)/Proton Antiporter Genes Involved in the Bacterial pH Homeostasis. International Journal of Molecular Sciences, 22(16), 8420.

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5

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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6

Ensuring High-Quality Ribosome Samples for NMR

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High resolution NMR experiments require that the sample of the macromolecular complex has to be pure from contaminating proteins and nucleic acids, it must be monodispersed and due to the experimental restraints of the technique highly concentrated. Purity of the final 30S ribosomes utilized in all subsequent experiments was judged by gel-based analysis in comparison to similarly purified 50S and 70S particles (Supplementary Fig. 8).
Ribosomes were further analyzed by Western Blot using a 6x-His Tag Antibody (Thermo Fischer) that allows selective detection of the modified ribosomal protein L12. The Western Blot with this specific antibody clearly shows that 30S particles are free of impurities of 50S protein components.
Integrity of ribosomes were further analyzed by native composite gel electrophoresis and quantitative mass spectrometry (Supplementary Fig. 9). After acquiring NMR experiments, the NMR samples were analyzed for RNA integrity and 30S ribosome stability. RNA integrity was checked by denaturing polyacrylamide gel electrophoresis (Supplementary Fig. 8). Theoretically, RNA could possibly be degraded by co-purification of RNases during ribosome preparation. Ribosome stability was further analyzed by loading the measured samples on another sucrose gradient (Supplementary Fig. 10).

de Jesus V., Qureshi N.S., Warhaut S., Bains J.K., Dietz M.S., Heilemann M., Schwalbe H, & Fürtig B. (2021). Switching at the ribosome: riboswitches need rProteins as modulators to regulate translation. Nature Communications, 12, 4723.

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7

SARS-CoV-2 Spike Protein Antibody Detection

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SARS-CoV-2 spike protein antibody is from GeneTex (GTX632604). ACE2 antibody is from GeneTex (GTX01160). 6x-His Tag antibody is from Thermo Fisher Scientific (MA1-21315-D550). GAPDH antibody is from OriGene (TA802519). HSC70 antibody is from Enzo (ADI-SPA-815-F). Clathrin heavy chain (CHC) antibody is from Cell Signaling Technology (4796S). Conjugated transferrin (Tf) antibody is from Thermo Fisher Scientific (T23366). Alexa Fluor 488, 568, and 647 conjugated secondary antibodies are from Invitrogen.

Bayati A., Kumar R., Francis V, & McPherson P.S. (2021). SARS-CoV-2 infects cells after viral entry via clathrin-mediated endocytosis. The Journal of Biological Chemistry, 296, 100306.

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8

Quantitative Protein Analysis by Western Blot

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The protein abundance analysis, Western blot was carried out. Whole cell lysis was separated on a 18% SDS-PAGE gel. Sample amount was equaled based on culture OD. Proteins on gel were transferred to a nitrocellulose membrane and detected by a primary 6x-His-tag Antibody (ThermoFisher, USA) from mouse and goat anti-mouse conjugated to alkaline phosphatase or HRP as a secondary antibody (Bio-Rad). Protein with His-tag on the nitrocellulose membrane was detected by using Opti-4CN kit (Bio-Rad). The intensity of western blot bands was analyzed using ImageJ.

Liu Y., Chen J., Khusnutdinova A.N., Correia K., Diep P., Batyrova K.A., Nemr K., Flick R., Stogios P., Yakunin A.F, & Mahadevan R. (2020). A novel C-terminal degron identified in bacterial aldehyde decarbonylases using directed evolution. Biotechnology for Biofuels, 13, 114.

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9

Bacterial Cell Wall Protein Binding Analysis

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A 20 ml of E. coli grown in LB medium, Clostridium thermocellum and C. cellulolyticum grown on 5 g/L cellobiose in VM medium were collected and centrifuged respectively. For each strain, the cell pellets were treated with NaN₃/Ca²⁺ and washed three times with 50 mM Phosphate-buffered saline (PBS) buffer (Sigma-Aldrich, St. Louis, MO, United States). Then, 50 μg purified X2-N, ΔX2-N, and CBM3a proteins were incubated with collected cell pellets respectively in the PBS buffer at 4°C for 12 h. After centrifugation, the pellet from each incubation was resuspended in the SDS-PAGE Protein Loading Buffer (Thermo Fisher, Waltham, MA, United States) and boiled for 10 min. Finally, the 6x-His Tag antibody (R930-25, Thermo Fisher, Waltham, MA, United States) was used to detect the binding between proteins and cell wall by Western blotting.

Tao X., Liu J., Kempher M.L., Xu T, & Zhou J. (2022). In vivo Functional Characterization of Hydrophilic X2 Modules in the Cellulosomal Scaffolding Protein. Frontiers in Microbiology, 13, 861549.

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10

Purification and characterization of Cas13a

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Cas13a was purified as referred to in previous studies (East-Seletsky et al., 2016 (link), 2017 (link); Su et al., 2020 (link)). Briefly, the Cas13a gene in plasmid pC019-LwCas13a from Leptotrichia wadei (Addgene, United States) was subcloned into the psmarti vector (xhoI restriction site), and the correct plasmid was transformed into BL21(DE3) (General Biosystem, China). The expression of the target protein was induced at different temperatures (15 and 37°C) and different concentrations of IPTG (0.2 and 1.0 mM) (Amresco, United States). The Cas13a protein was purified by Ni-NTA (Smart-Lifesciences, China) using the 6 × His Tag antibody and horseradish peroxidase conjugate (Invitrogen, United States). After the addition of SUMO Protease (General Biosystem, China) to remove the fusion SUMO label, and the purified target protein was dialyzed into protein buffer [50 mM Tris, pH 7.5, 600 mM NaCl, 5% (vol/vol) glycerol, and 2 mM DTT].

An B., Zhang H., Su X., Guo Y., Wu T., Ge Y., Zhu F, & Cui L. (2021). Rapid and Sensitive Detection of Salmonella spp. Using CRISPR-Cas13a Combined With Recombinase Polymerase Amplification. Frontiers in Microbiology, 12, 732426.

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His tag antibody

Lab products found in correlation

15 protocols using his tag antibody

Purification and Glycosylation Analysis of Recombinant Proteins

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

Detecting Recombinant Protein Expression

Immunodetection of E. chaffeensis ECH_0379 Protein

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

Ensuring High-Quality Ribosome Samples for NMR

SARS-CoV-2 Spike Protein Antibody Detection

Quantitative Protein Analysis by Western Blot

Bacterial Cell Wall Protein Binding Analysis

Purification and characterization of Cas13a

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