10k mwco centrifugal filters

Manufactured by Merck Group

The 10K MWCO centrifugal filters are a lab equipment product designed for molecular weight cut-off (MWCO) filtration. These filters are used to separate molecules or particles based on their molecular weight, allowing the passage of smaller molecules while retaining larger ones.

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Lab products found in correlation

Ni sepharose 6 ff column, by Cytiva (6 mentions) Akta avant150 fplc system, by Cytiva (6 mentions) Superdex 200 increase 10 300, by Cytiva (5 mentions) Hiload s200 pg gl sec column, by Cytiva (5 mentions) Bioflo 320 fermenter, by Eppendorf (4 mentions) Superdex 200 increase 10 300 sec column, by Cytiva (1 mentions) Anti ha affinity matrix, by Roche (1 mentions) Ha peptide, by Roche (1 mentions)

8 protocols using 10k mwco centrifugal filters

Purification of I53-50A and I53-50B Proteins

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The I53-50A and I53-50B proteins were expressed as described before (Walls et al., 2020b (link)). Briefly, transformed Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) were grown at 37°C to an OD600 ∼0.8 with agitation. Expression was induced with 1 mM IPTG and temperature was reduced to 18°C. Cells were harvested after ∼16 h and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Proteins were eluted with a linear gradient of 30 mM to 500 mM imidazole in 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) previously equilibrated in 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer.

Tortorici M.A., Walls A.C., Joshi A., Park Y.J., Eguia R.T., Miranda M.C., Kepl E., Dosey A., Stevens-Ayers T., Boeckh M.J., Telenti A., Lanzavecchia A., King N.P., Corti D., Bloom J.D, & Veesler D. (2022). Structure, receptor recognition, and antigenicity of the human coronavirus CCoV-HuPn-2018 spike glycoprotein. Cell, 185(13), 2279-2291.e17.

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Purification of I53-50A and I53-50B.4PT1 Proteins

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The I53–50A and I53–50B.4.PT1 proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ~0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ~16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6×10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53–50A elutes at ~0.6 column volume (CV). I53–50B.4PT1 elutes at ~0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Walls A.C., Fiala B., Schäfer A., Wrenn S., Pham M.N., Murphy M., Tse L.V., Shehata L., O’Connor M.A., Chen C., Navarro M.J., Miranda M.C., Pettie D., Ravichandran R., Kraft J.C., Ogohara C., Palser A., Chalk S., Lee E.C., Kepl E., Chow C.M., Sydeman C., Hodge E.A., Brown B., Fuller J.T., Dinnon KH I.I.I., Gralinski L.E., Leist S.R., Gully K.L., Lewis T.B., Guttman M., Chu H.Y., Lee K.K., Fuller D.H., Baric R.S., Kellam P., Carter L., Pepper M., Sheahan T.P., Veesler D, & King N.P. (2020). Elicitation of potent neutralizing antibody responses by designed protein nanoparticle vaccines for SARS-CoV-2. bioRxiv.

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Purification and Characterization of I53-50A and I53-50B.4PT1 Proteins

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The I53-50A and I53-50B.4.PT1 proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ∼0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ∼0.6 column volume (CV). I53-50B.4PT1 elutes at ∼0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Walls A.C., Fiala B., Schäfer A., Wrenn S., Pham M.N., Murphy M., Tse L.V., Shehata L., O’Connor M.A., Chen C., Navarro M.J., Miranda M.C., Pettie D., Ravichandran R., Kraft J.C., Ogohara C., Palser A., Chalk S., Lee E.C., Guerriero K., Kepl E., Chow C.M., Sydeman C., Hodge E.A., Brown B., Fuller J.T., Dinnon KH I.I.I., Gralinski L.E., Leist S.R., Gully K.L., Lewis T.B., Guttman M., Chu H.Y., Lee K.K., Fuller D.H., Baric R.S., Kellam P., Carter L., Pepper M., Sheahan T.P., Veesler D, & King N.P. (2020). Elicitation of Potent Neutralizing Antibody Responses by Designed Protein Nanoparticle Vaccines for SARS-CoV-2. Cell, 183(5), 1367-1382.e17.

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Purification of Nanoparticle Components

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The nanoparticle components I53-50A and I53-50B.4.PT1,²⁹ (link) and I53_dn5A and I53_dn5B,³⁰ (link) were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) and grown in 2 L baffled shake flasks. Cells were grown at 37°C to an OD600–0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells were shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, (with 0.75% CHAPS only for I53-50 proteins). Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM–500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, (with 0.75% CHAPS only for I53-50 proteins) buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to a Superdex 200 Increase 10/300 SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, (with 0.75% CHAPS only for I53-50 proteins) buffer. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Kraft J.C., Pham M.N., Shehata L., Brinkkemper M., Boyoglu-Barnum S., Sprouse K.R., Walls A.C., Cheng S., Murphy M., Pettie D., Ahlrichs M., Sydeman C., Johnson M., Blackstone A., Ellis D., Ravichandran R., Fiala B., Wrenn S., Miranda M., Sliepen K., Brouwer P.J., Antanasijevic A., Veesler D., Ward A.B., Kanekiyo M., Pepper M., Sanders R.W, & King N.P. (2022). Antigen- and scaffold-specific antibody responses to protein nanoparticle immunogens. Cell Reports Medicine, 3(10), 100780.

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Expression and Purification of I53-50A and I53-50B.4PT1

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The I53-50A and I53-50B.4.PT1 proteins (Bale et al., 2016 (link)) were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ∼0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50A elutes at ∼0.6 column volume (CV). I53-50B.4PT1 elutes at ∼0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

Walls A.C., Miranda M.C., Schäfer A., Pham M.N., Greaney A., Arunachalam P.S., Navarro M.J., Tortorici M.A., Rogers K., O’Connor M.A., Shirreff L., Ferrell D.E., Bowen J., Brunette N., Kepl E., Zepeda S.K., Starr T., Hsieh C.L., Fiala B., Wrenn S., Pettie D., Sydeman C., Sprouse K.R., Johnson M., Blackstone A., Ravichandran R., Ogohara C., Carter L., Tilles S.W., Rappuoli R., Leist S.R., Martinez D.R., Clark M., Tisch R., O’Hagan D.T., Van Der Most R., Van Voorhis W.C., Corti D., McLellan J.S., Kleanthous H., Sheahan T.P., Smith K.D., Fuller D.H., Villinger F., Bloom J., Pulendran B., Baric R.S., King N.P, & Veesler D. (2021). Elicitation of broadly protective sarbecovirus immunity by receptor-binding domain nanoparticle vaccines. Cell, 184(21), 5432-5447.e16.

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Mutagenesis and Expression of HDAC8

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The G302A, G303A, G304A, and G305A mutations were introduced into the human HDAC8-His₆-pET20b construct^{11 (link)} using Quickchange site-directed mutagenesis (Agilent Technologies, Inc.). Forward and reverse primers were synthesized by Integrated DNA Technologies (Table 1). PCR plasmid products were sequenced to confirm mutations. HDAC8 constructs were expressed in BL21(DE3) Escherichia coli cells as previously described,^{11 (link)} with minor modifications. Briefly, 50 mL cultures (Lysogeny Broth (LB) at 100 μg/mL ampicillin) were grown overnight and used to inoculate 12 × 1-L M9 minimal media supplemented with 100 μg/mL ampicillin. Cells were grown at 37 °C until OD₆₀₀ reached ~1.0, at which point the temperature was lowered to 16 °C. After cooling for 30 min, expression was induced with 100 μM isopropyl-β-thiogalactopyranoside and 100 μM ZnCl₂. Protein was expressed overnight. Cells were pelleted via centrifugation and stored at −80 °C until purification. Wild-type and mutant constructs of human HDAC8 were purified as previously described.^{11 (link)} All protein was concentrated via centrifugation using 10k MWCO centrifugal filters (Millipore) to 10–20 mg/mL. Protein concentrations were determined from the absorbance at 280 nm using the calculated extinction coefficient 50,240 M⁻¹cm⁻¹. Aliquots were flash-cooled in liquid nitrogen and stored at −80 °C until further use.

Porter N.J., Christianson N.H., Decroos C, & Christianson D.W. (2016). Structural and Functional Influence of the Glycine-Rich Loop G302GGGY on the Catalytic Tyrosine of Histone Deacetylase 8. Biochemistry, 55(48), 6718-6729.

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Purification of I53-50B.4PT1 and 2obx Proteins

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The I53-50B.4.PT1 and 2obx proteins were expressed in Lemo21(DE3) (NEB) in LB (10 g Tryptone, 5 g Yeast Extract, 10 g NaCl) grown in 2 L baffled shake flasks or a 10 L BioFlo 320 Fermenter (Eppendorf). Cells were grown at 37°C to an OD600 ∼0.8, and then induced with 1 mM IPTG. Expression temperature was reduced to 18°C and the cells shaken for ∼16 h. The cells were harvested and lysed by microfluidization using a Microfluidics M110P at 18,000 psi in 50 mM Tris, 500 mM NaCl, 30 mM imidazole, 1 mM PMSF, 0.75% CHAPS. Lysates were clarified by centrifugation at 24,000 g for 30 min and applied to a 2.6 × 10 cm Ni Sepharose 6 FF column (Cytiva) for purification by IMAC on an AKTA Avant150 FPLC system (Cytiva). Protein of interest was eluted over a linear gradient of 30 mM to 500 mM imidazole in a background of 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. Peak fractions were pooled, concentrated in 10K MWCO centrifugal filters (Millipore), sterile filtered (0.22 μm) and applied to either a Superdex 200 Increase 10/300, or HiLoad S200 pg GL SEC column (Cytiva) using 50 mM Tris pH 8, 500 mM NaCl, 0.75% CHAPS buffer. I53-50B.4PT1 and 2obx elute at ∼0.45 CV. After sizing, bacterial-derived components were tested to confirm low levels of endotoxin before using for nanoparticle assembly.

McLeod B., Mabrouk M.T., Miura K., Ravichandran R., Kephart S., Hailemariam S., Pham T.P., Semesi A., Kucharska I., Kundu P., Huang W.C., Johnson M., Blackstone A., Pettie D., Murphy M., Kraft J.C., Leaf E.M., Jiao Y., van de Vegte-Bolmer M., van Gemert G.J., Ramjith J., King C.R., MacGill R.S., Wu Y., Lee K.K., Jore M.M., King N.P., Lovell J.F, & Julien J.P. (2022). Vaccination with a structure-based stabilized version of malarial antigen Pfs48/45 elicits ultra-potent transmission-blocking antibody responses. Immunity, 55(9), 1680-1692.e8.

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Optimized Purification of HA-tagged Proteins

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Transfection of FreeStyle™ 293 cells was done according to the manufacturer's instruction. The culture supernatants were harvested at 72h post transfection and filtered through 0.45μm filter unit before purification. The supernatants were concentrated with 10k MWCO centrifugal filters (Millipore) and dialyzed with PBS before further purification. Small-scale protein purification was performed according to the manufacturer's protocol for anti-HA affinity matrix (Roche). Briefly, samples were incubated with anti-HA affinity matrix and following three washes, specifically-bound proteins were eluted by HA peptide (Roche) in elution buffer and then dialyzed with PBS three times. The purity of each protein was analyzed using coomassie blue staining, Silverquest staining (Invitrogen) and SEC analysis.

Li C., Wang J., Hu J., Feng Y., Hasegawa K., Peng X., Duan X., Zhao A., Mikitsh J.L., Muzykantov V.R., Chacko A.M., Pryma D.A., Dunn S.M, & Coukos G. (2014). Development, optimization, and validation of novel anti-TEM1/CD248 affinity agent for optical imaging in cancer. Oncotarget, 5(16), 6994-7012.

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