Amicon 10 000 kda mwco filtration unit

Manufactured by Merck Group

The Amicon 10,000 KDa MWCO filtration unit is a laboratory equipment designed for the separation and concentration of macromolecules such as proteins, enzymes, and other high-molecular-weight compounds. The device utilizes a semi-permeable membrane with a molecular weight cutoff (MWCO) of 10,000 Daltons, allowing the passage of smaller molecules while retaining the target macromolecules. This filtration unit is commonly used in various research and development applications that require sample preparation, purification, and concentration.

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Lab products found in correlation

Freestyle media, by Thermo Fisher Scientific (2 mentions) Hitrap ninta column, by GE Healthcare (2 mentions) Superdex 200 16 600 column, by GE Healthcare (2 mentions) Ni nta, by Qiagen (2 mentions) Superdex 75 16 600 column, by GE Healthcare (1 mentions)

Spelling variants of this product

Filtration unit (11 citations) MWCO-10,000 (8 citations) MWCO 10 kDa (6 citations) Amicon 10 kDa MWCO filtration unit (2 citations)

4 protocols using amicon 10 000 kda mwco filtration unit

Recombinant VHH Protein Production

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The B3-, and VHH control 1B7-IFNg coding sequences were sub-cloned into the mammalian expression vector pVRC and transiently transfected using polyethyleneimine into HEK293F cells cultured in FreeStyle media (ThermoFisher #12338018). Media containing secreted protein was harvested 6 days following transfection by centrifugation at 8000 g for 20 minutes at 4°C, then loaded onto a HiTrap NiNTA column (GE Healthcare) and washed with 50 mM Tris, pH 8, 150 mM NaCl and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole and 10% glycerol, then loaded onto a Superdex 200 16/600 column (GE Healthcare) in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. Recombinant VHH purity was assessed by SDS-PAGE, and peak fractions were recovered and concentrated with an Amicon 10,000 KDa MWCO filtration unit (Millipore), and stored at −80°C.

Lenehan P.J., Cirella A., Uchida A.M., Crowley S.J., Sharova T., Boland G., Dougan M., Dougan S.K, & Heckler M. (2021). Type 2 immunity is maintained during cancer-associated adipose tissue wasting. Immunotherapy Advances, 1(1), ltab011.

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Periplasmic Expression and Purification of VHH

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B3 coding sequence were sub-cloned into the E. coli periplasmic expression vector pHEN6 using the PCR primers 189 (5′-tactcgcggcccagccGGCCCAACCGGCCATGGC-3′) and 190 (5′-agtcctcctgaggagacggtgaccGAGACGGTGACCTGGGTCCCC-3′) to allow for Gibson cloning and the inclusion of a C-terminal sortase motif and 6xHis tag. WK6 E. coli containing the plasmid were grown to mid-log phase at 37 °C in TB plus ampicillin, and induced with 1 mM IPTG overnight at 30 °C. Cells were harvested by centrifugation at 5000 × g for 15 min at 4 °C, then resuspened in 25 ml 1x TES buffer (200 mM Tris, pH 8, 0.65 mM EDTA, 0.5 M sucrose) per liter culture, and incubated for 1 h at 4 °C with agitation. Resuspended cells wre then submitted to osmotic shock by diluting 1:4 in 0.25× TES, and incubating overnight at 4 °C. The periplasmic fraction was isolated by centrifugation at 8000 rpm for 30 min at 4 °C, and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole and 10% glycerol, then loaded onto a Superdex 75 10/300 column in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. Recombinant VHH purity was assessed by SDS-PAGE, and peak fractions were recovered and concentrated with an Amicon 10,000 KDa MWCO filtration unit (Millipore), and stored at −80 °C.

Ingram J.R., Dougan M., Rashidian M., Knoll M., Keliher E.J., Garrett S., Garforth S., Blomberg O.S., Espinosa C., Bhan A., Almo S.C., Weissleder R., Lodish H., Dougan S.K, & Ploegh H.L. (2017). PD-L1 is an activation-independent marker of brown adipocytes. Nature Communications, 8, 647.

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Expression and Purification of PD-L1 VHH

Cited 1 time

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B3 and A12 are specific for PD-L1 as described (3 (link)). The B3-IL2 coding sequence was subcloned into the E.coli periplasmic expression vector pET22b, with the inclusion of a C-terminal sortase motif and His₆ tag. BL21(DE3) E. coli containing the plasmid was grown to mid-log phase at 37°C in TB plus ampicillin, and induced with 0.5 mM IPTG overnight at 25°C. Cells were harvested by centrifugation at 5000×g for 15 minutes at 4°C, then resuspended in 25 ml 1x TES buffer (200 mM Tris, pH 8, 0.65 mM EDTA, 0.5 M sucrose) per liter culture, and incubated for 1 hour at 4°C with agitation. Resuspended cells were then submitted to osmotic shock by diluting 1:4 in 0.25x TES, and incubating overnight at 4°C. The periplasmic fraction was isolated by centrifugation at 8000rpm for 30 min at 4°C, and then loaded onto Ni-NTA (Qiagen) in 50 mM Tris, pH 8, 150 mM NaCl and 10 mM imidazole. Protein was eluted in 50 mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole and 10% glycerol, then loaded onto a Superdex 75 16/600 column (GE Healthcare) in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. Recombinant VHH purity was assessed by SDS-PAGE, and peak fractions were recovered and concentrated with an Amicon 10,000 KDa MWCO filtration unit (Millipore), and stored at −80°C.

Dougan M., Ingram J.R., Jeong H.J., Mosaheb M.M., Bruck P.T., Ali L., Pishesha N., Blomberg O., Tyler P.M., Servos M.M., Rashidian M., Nguyen Q.D., von Andrian U.H., Ploegh H.L, & Dougan S.K. (2018). Targeting cytokine therapy to the pancreatic tumor microenvironment using PD-L1 specific VHHs. Cancer immunology research, 6(4), 389-401.

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Expression and Purification of VHH Proteins

Cited 1 time

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The PDL1 specific VHH A12-, B3-, and irrelevant specificity VHHCTR(1B7)-IFNγ coding sequences were subcloned into the mammalian expression vector pVRC and transiently transfected using polyethyleneimine into HEK293F cells cultured in Freestyle media (ThermoFisher) (3 (link)). Media containing secreted protein was harvested six days following transfection by centrifugation at 8,000 × g for 20 min at 4°C, then loaded onto a HiTrap NiNTA column (GE Healthcare) and washed with 50 mM Tris, pH 8, 150 mM NaCl and 10 mM imidazole. Protein was eluted in 50- mM Tris, pH 8, 150 mM NaCl, 500 mM imidazole and 10% glycerol, then loaded onto a Superdex 200 16/600 column (GE Healthcare) in 50 mM Tris, pH 8, 150 mM NaCl, 10% glycerol. Recombinant VHH purity was assessed by SDS-PAGE, and peak fractions were recovered and concentrated with an Amicon 10,000 KDa MWCO filtration unit (Millipore), and stored at −80°C.

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