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centrifuge 5425 with rotor fa 24 × 2  (Eppendorf AG)


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    Eppendorf AG centrifuge 5425 with rotor fa 24 × 2
    Centrifuge 5425 With Rotor Fa 24 × 2, supplied by Eppendorf AG, used in various techniques. Bioz Stars score: 96/100, based on 11 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/product/rotor+fa+24x2/Rotor+FA-24x2/pmc13123351-24-0-6
    Average 96 stars, based on 11 article reviews
    centrifuge 5425 with rotor fa 24 × 2 - by Bioz Stars, 2026-09
    96/100 stars

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    Related Articles

    Centrifugation:

    Article Title: YebC2 resolves ribosome stalling and increases fitness of cells lacking EF-P and the ABCF ATPase YfmR
    Article Snippet: .. Cells were lysed using a homogenizer (Beadbug6, Benchmark) by five 20 second pulses at speed 4350 rpm with chilling on ice for 2 min between the cycles and clarified by centrifugation at 21,300 rcf for 20 min (Eppendorf 5425R, rotor FA-24x2). ..

    Article Title: YebC2 resolves ribosome stalling at polyprolines independent of EF-P and the ABCF ATPase YfmR
    Article Snippet: .. Cells lysed using a homogenizer (Beadbug6, Benchmark) by five 20 second pulses at speed 4350 rpm with chilling on ice for 2 min between the cycles and clarified by centrifugation at 21,300 rcf for 20 min (Eppendorf 5425R, rotor FA-24x2). ..

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: .. Day 9 – (1) Filter HPLC IMAC elution into a centrifugal concentrator (Vivaspin 20 mL MWCO 10 kDa a ); (2) centrifuge (Eppendorf 5920 R, rotor FA-6x50, 6000 xg b , 4°C) to concentrate sample to ≤1 mL but >0.5 mL; (3) add HSB to concentrator to 14 mL mark for a >10-fold dilution into HSB; (4) repeat steps (2-3) three times for >38,416-fold total dilution (i.e., BE) of the original buffer (IMAC Buffer: 75% A, 25% B) to reduce imidazole to ≤6.5 μM; (5) repeat step (2); (6) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (7) measure sample A 280 (DeNovix DS-11 FX+) to calculate 8His-TEV-RoxP (MDH..ALN) concentration (ɛ280 14565 M -1 cm -1 , MW 17228.04 g/mol) and yield c ; and (8) then store RoxP protein sample at 4°C. a RoxP_1 was retained by MWCO 10 kDa, but two RoxP orthologs (e.g., RoxP_8, _11) were not retained by this cutoff and required MWCO 3 kDa to be concentrated. b GE Healthcare Vivaspin centrifugal concentrators were used for the original gravity-flow IMAC work, and GE recommended a maximum centrifugation speed (× g) of 8000 xg. .. When that protocol was adapted to an HPLC approach, Sartorius was then the distributor of these centrifugal concentrators, and Sartorius recommended a maximum centrifugation speed (× g) of 6000 xg c Expected 8His_TEV_RoxP_1 yield after BE: ≥60 mg from a 500 mL autoinduction culture.

    Article Title:
    Article Snippet: Equal volumes of gradient fractions were separated by SDS-PAGE and probed with an anti-Flag antibody (Sigma A8592) or with polyclonal antibody raised against B. subtilis EF-Tu (8). .. The cell resuspension was lysed using a homogenizer (Beadbug6, Benchmark) by four 30 s pulses at speed 4350 rpm with chilling on ice for 3 min between the cycles and clarified by centrifugation at 21,300 rcf for 20 min (Eppendorf 5425R, rotor FA-24x2). ..

    Article Title: YebC2 resolves ribosome stalling and increases fitness of cells lacking EF-P and the ABCF ATPase YfmR.
    Article Snippet: .. Cells were lysed using a homogenizer (Beadbug6, Benchmark) by five 20 second pulses at speed 4350 rpm with chilling on ice for 2 min between the cycles and clarified by centrifugation at 21,300 rcf for 20 min (Eppendorf 5425R, rotor FA-24x2). ..

    Protein Concentration:

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: Day 10 – (1) Hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (2) repeat “ Buffer-Exchange (BE). .. Day 9 – (7) ” above to assess soluble protein concentration in supernatant b ; (3) add 1/9 sample volume (31.4 μL) of 10x IMAC-BB and 0.63 μL of 5.0 M imidazole (pH 7.8) to your sample to adjust chemical/salt concentrations to 16 mM HEPES, 420 mM NaCl, 0.008% azide, 50 m Na phosphate, 10 mM imidazole, pH ∼8; (4) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (5) remove His-tagged proteins from sample using one His-Spin Protein Miniprep (Zymo Research) and collect His-Spin FT containing RoxP-tagless; (6) perform BE into HSB using 2.0 mL Zeba Desalting Column (Thermo Scientific); (7) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (8) measure sample A 280 (DeNovix DS-11 FX+) to calculate the RoxP-tagless (GAT..ALN) concentration (ɛ280 13075 M -1 cm -1 , MW 14888.59 g/mol); (9) store RoxP protein sample at 4°C; and (10) verify BE and tag removal samples by SDS PAGE. a Reaction volume was scaled up as dictated by RoxP-tagless needs of planned experiments. b Only an estimation, as cleaved N-terminal tag is less soluble (i.e., may be in the post-centrifugation pellet) and a small amount of NEB TEV protease is present, though it is likely ≤5 μg per reaction. ..

    Concentration Assay:

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: Day 10 – (1) Hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (2) repeat “ Buffer-Exchange (BE). .. Day 9 – (7) ” above to assess soluble protein concentration in supernatant b ; (3) add 1/9 sample volume (31.4 μL) of 10x IMAC-BB and 0.63 μL of 5.0 M imidazole (pH 7.8) to your sample to adjust chemical/salt concentrations to 16 mM HEPES, 420 mM NaCl, 0.008% azide, 50 m Na phosphate, 10 mM imidazole, pH ∼8; (4) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (5) remove His-tagged proteins from sample using one His-Spin Protein Miniprep (Zymo Research) and collect His-Spin FT containing RoxP-tagless; (6) perform BE into HSB using 2.0 mL Zeba Desalting Column (Thermo Scientific); (7) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (8) measure sample A 280 (DeNovix DS-11 FX+) to calculate the RoxP-tagless (GAT..ALN) concentration (ɛ280 13075 M -1 cm -1 , MW 14888.59 g/mol); (9) store RoxP protein sample at 4°C; and (10) verify BE and tag removal samples by SDS PAGE. a Reaction volume was scaled up as dictated by RoxP-tagless needs of planned experiments. b Only an estimation, as cleaved N-terminal tag is less soluble (i.e., may be in the post-centrifugation pellet) and a small amount of NEB TEV protease is present, though it is likely ≤5 μg per reaction. ..

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: .. Day 9 – (1) Filter HPLC IMAC elution into a centrifugal concentrator (Vivaspin 20 mL MWCO 10 kDa a ); (2) centrifuge (Eppendorf 5920 R, rotor FA-6x50, 6000 xg b , 4°C) to concentrate sample to ≤1 mL but >0.5 mL; (3) add HSB to concentrator to 14 mL mark for a >10-fold dilution into HSB; (4) repeat steps (2-3) three times for >38,416-fold total dilution (i.e., BE) of the original buffer (IMAC Buffer: 75% A, 25% B) to reduce imidazole to ≤6.5 μM; (5) repeat step (2); (6) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (7) measure sample A 280 (DeNovix DS-11 FX+) to calculate 8His-TEV-RoxP (MDH..ALN) concentration (ɛ280 14565 M -1 cm -1 , MW 17228.04 g/mol) and yield c ; and (8) then store RoxP protein sample at 4°C. a RoxP_1 was retained by MWCO 10 kDa, but two RoxP orthologs (e.g., RoxP_8, _11) were not retained by this cutoff and required MWCO 3 kDa to be concentrated. b GE Healthcare Vivaspin centrifugal concentrators were used for the original gravity-flow IMAC work, and GE recommended a maximum centrifugation speed (× g) of 8000 xg. .. When that protocol was adapted to an HPLC approach, Sartorius was then the distributor of these centrifugal concentrators, and Sartorius recommended a maximum centrifugation speed (× g) of 6000 xg c Expected 8His_TEV_RoxP_1 yield after BE: ≥60 mg from a 500 mL autoinduction culture.

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: Day 4 – (1) Incubate 10.0 mL of B-PER Complete Protein Extraction (stored at 4°C) in a ≥100 mL RT aluminum bead bath for ≥20 min to raise the temperature of this reagent to RT; (2) Incubate 25.0 mL of NiNTA-WB in an ice-bath for ≥30 min to prechill it to 4°C; (3) spin down 70 mL of ZYM-5052-Carb autoinduction culture (Eppendorf 5920 R, rotor FA-6x50; 10000 x g, 10 min, 4°C); (4) mass the cell pellet a ; (5) prepare 25 mL of RT B-PER with protease inhibitors (BPER-PI) b ; (6) collect cells by centrifugation (Eppendorf 5920 R, rotor FA-6x50, 10000 xg, 10 min, 4°C); (7) freeze-thaw the cell pellet (-20°C for 30 min then RT until thawed) c ; (8) gently resuspend cell pellet in BPER-PI and incubate with gentle inversion at RT for 15 min; (9) centrifuge lysate (Eppendorf 5920 R, rotor FA-6x50; 16000 x g, 20 min, 4°C) and transfer supernatant to a new 50 mL conical; (10) add 10 mL of ice-cold NiNTA-WB to lysate supernatant (1:1 lysate:buffer volume; imidazole final concentration: 10 mM; non-EDTA protease inhibitor final concentration: 1x); (11) filter sample; (12) concentrate to 2.0-4.0 mL using a Vivaspin 20 mL MWCO 10 d ; (13) hard spin sample (Eppendorf 5425 R, rotor FA-2x24; 21300 x g, 10 min, 4°C); (14) filter sample; (15) purify 8His-TEV-RoxP using one His-Spin Protein Miniprep column (Zymo Research) according to manufacturer’s instructions; (16) use a 2.0 mL Zeba Desalting Column (Thermo Scientific) to BE the His-Spin elution into PBS; (17) measure sample A 280 (DeNovix DS-11 FX+) to calculate 8His-TEV-RoxP concentration (MDH..ALN, ɛ280 14565 M -1 cm -1 , MW 17228.04 g/mol) e and yield f ; (18) add 26.7 μL of 10x NEB TEV Protease Reaction Buffer; (19) add 4 μL (40 units) of NEB TEV Protease Enzyme; (20) place sample in a tube rotator at 4°C, and (21) gently rotate sample overnight (≥16 hr). .. Day 5 – (1) Add 1/9 sample volume (30.1 μL) of 10x IMAC-BB and 0.60 μL of 5.0 M imidazole (pH 7.8) to the sample to adjust chemical/salt concentrations to 16 mM HEPES, 420 mM NaCl, 0.008% azide, 50 m Na phosphate, 10 mM imidazole, pH ∼8; (2) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (3) remove His-tagged proteins from sample using one His-Spin Protein Miniprep and collect His-Spin FT containing RoxP-tagless; (4) perform BE into HSB using 2.0 mL Zeba Desalting Column; (5) measure sample A 280 (DeNovix DS-11 FX+) to calculate the RoxP-tagless concentration (GAT..ALN, ɛ280 13075 M -1 cm -1 , MW 14888.59 g/mol) i and yield j ; (6) store RoxP protein sample at 4°C; and (7) verify BE and tag removal samples by SDS PAGE k . a Average cell pellet yield from 70 mL of autoinduction culture grown overnight at 18°C was 1.96 g, which is 78% of the cell pellet yield expected based on the larger scale (500 mL), 5-day at 18°C autoinduction culture. b A 10.0 mL aliquot of RT B-PER Complete Protein Extraction Reagent was supplemented with protease inhibitors aprotinin (4.0 μg/mL), leupeptin (40.0 μM), Pepstatin A (4.0 μM), PMSF (2.0 mM), and EDTA pH 8.0 (2.0 mM). ..

    SDS Page:

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: Day 10 – (1) Hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (2) repeat “ Buffer-Exchange (BE). .. Day 9 – (7) ” above to assess soluble protein concentration in supernatant b ; (3) add 1/9 sample volume (31.4 μL) of 10x IMAC-BB and 0.63 μL of 5.0 M imidazole (pH 7.8) to your sample to adjust chemical/salt concentrations to 16 mM HEPES, 420 mM NaCl, 0.008% azide, 50 m Na phosphate, 10 mM imidazole, pH ∼8; (4) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (5) remove His-tagged proteins from sample using one His-Spin Protein Miniprep (Zymo Research) and collect His-Spin FT containing RoxP-tagless; (6) perform BE into HSB using 2.0 mL Zeba Desalting Column (Thermo Scientific); (7) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (8) measure sample A 280 (DeNovix DS-11 FX+) to calculate the RoxP-tagless (GAT..ALN) concentration (ɛ280 13075 M -1 cm -1 , MW 14888.59 g/mol); (9) store RoxP protein sample at 4°C; and (10) verify BE and tag removal samples by SDS PAGE. a Reaction volume was scaled up as dictated by RoxP-tagless needs of planned experiments. b Only an estimation, as cleaved N-terminal tag is less soluble (i.e., may be in the post-centrifugation pellet) and a small amount of NEB TEV protease is present, though it is likely ≤5 μg per reaction. ..

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: Day 4 – (1) Incubate 10.0 mL of B-PER Complete Protein Extraction (stored at 4°C) in a ≥100 mL RT aluminum bead bath for ≥20 min to raise the temperature of this reagent to RT; (2) Incubate 25.0 mL of NiNTA-WB in an ice-bath for ≥30 min to prechill it to 4°C; (3) spin down 70 mL of ZYM-5052-Carb autoinduction culture (Eppendorf 5920 R, rotor FA-6x50; 10000 x g, 10 min, 4°C); (4) mass the cell pellet a ; (5) prepare 25 mL of RT B-PER with protease inhibitors (BPER-PI) b ; (6) collect cells by centrifugation (Eppendorf 5920 R, rotor FA-6x50, 10000 xg, 10 min, 4°C); (7) freeze-thaw the cell pellet (-20°C for 30 min then RT until thawed) c ; (8) gently resuspend cell pellet in BPER-PI and incubate with gentle inversion at RT for 15 min; (9) centrifuge lysate (Eppendorf 5920 R, rotor FA-6x50; 16000 x g, 20 min, 4°C) and transfer supernatant to a new 50 mL conical; (10) add 10 mL of ice-cold NiNTA-WB to lysate supernatant (1:1 lysate:buffer volume; imidazole final concentration: 10 mM; non-EDTA protease inhibitor final concentration: 1x); (11) filter sample; (12) concentrate to 2.0-4.0 mL using a Vivaspin 20 mL MWCO 10 d ; (13) hard spin sample (Eppendorf 5425 R, rotor FA-2x24; 21300 x g, 10 min, 4°C); (14) filter sample; (15) purify 8His-TEV-RoxP using one His-Spin Protein Miniprep column (Zymo Research) according to manufacturer’s instructions; (16) use a 2.0 mL Zeba Desalting Column (Thermo Scientific) to BE the His-Spin elution into PBS; (17) measure sample A 280 (DeNovix DS-11 FX+) to calculate 8His-TEV-RoxP concentration (MDH..ALN, ɛ280 14565 M -1 cm -1 , MW 17228.04 g/mol) e and yield f ; (18) add 26.7 μL of 10x NEB TEV Protease Reaction Buffer; (19) add 4 μL (40 units) of NEB TEV Protease Enzyme; (20) place sample in a tube rotator at 4°C, and (21) gently rotate sample overnight (≥16 hr). .. Day 5 – (1) Add 1/9 sample volume (30.1 μL) of 10x IMAC-BB and 0.60 μL of 5.0 M imidazole (pH 7.8) to the sample to adjust chemical/salt concentrations to 16 mM HEPES, 420 mM NaCl, 0.008% azide, 50 m Na phosphate, 10 mM imidazole, pH ∼8; (2) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (3) remove His-tagged proteins from sample using one His-Spin Protein Miniprep and collect His-Spin FT containing RoxP-tagless; (4) perform BE into HSB using 2.0 mL Zeba Desalting Column; (5) measure sample A 280 (DeNovix DS-11 FX+) to calculate the RoxP-tagless concentration (GAT..ALN, ɛ280 13075 M -1 cm -1 , MW 14888.59 g/mol) i and yield j ; (6) store RoxP protein sample at 4°C; and (7) verify BE and tag removal samples by SDS PAGE k . a Average cell pellet yield from 70 mL of autoinduction culture grown overnight at 18°C was 1.96 g, which is 78% of the cell pellet yield expected based on the larger scale (500 mL), 5-day at 18°C autoinduction culture. b A 10.0 mL aliquot of RT B-PER Complete Protein Extraction Reagent was supplemented with protease inhibitors aprotinin (4.0 μg/mL), leupeptin (40.0 μM), Pepstatin A (4.0 μM), PMSF (2.0 mM), and EDTA pH 8.0 (2.0 mM). ..

    High Performance Liquid Chromatography:

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: .. Day 9 – (1) Filter HPLC IMAC elution into a centrifugal concentrator (Vivaspin 20 mL MWCO 10 kDa a ); (2) centrifuge (Eppendorf 5920 R, rotor FA-6x50, 6000 xg b , 4°C) to concentrate sample to ≤1 mL but >0.5 mL; (3) add HSB to concentrator to 14 mL mark for a >10-fold dilution into HSB; (4) repeat steps (2-3) three times for >38,416-fold total dilution (i.e., BE) of the original buffer (IMAC Buffer: 75% A, 25% B) to reduce imidazole to ≤6.5 μM; (5) repeat step (2); (6) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (7) measure sample A 280 (DeNovix DS-11 FX+) to calculate 8His-TEV-RoxP (MDH..ALN) concentration (ɛ280 14565 M -1 cm -1 , MW 17228.04 g/mol) and yield c ; and (8) then store RoxP protein sample at 4°C. a RoxP_1 was retained by MWCO 10 kDa, but two RoxP orthologs (e.g., RoxP_8, _11) were not retained by this cutoff and required MWCO 3 kDa to be concentrated. b GE Healthcare Vivaspin centrifugal concentrators were used for the original gravity-flow IMAC work, and GE recommended a maximum centrifugation speed (× g) of 8000 xg. .. When that protocol was adapted to an HPLC approach, Sartorius was then the distributor of these centrifugal concentrators, and Sartorius recommended a maximum centrifugation speed (× g) of 6000 xg c Expected 8His_TEV_RoxP_1 yield after BE: ≥60 mg from a 500 mL autoinduction culture.

    Buffer Exchange:

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: Day 9 – (1) Prepare 250 μL of 2.0 mg/mL RoxP in HSB a ; (2) add 28.2 μL of 10x NEB TEV Protease Reaction Buffer (0.5 M Tris-HCl pH 7.5, 5.0 mM EDTA, 10.0 mM DTT); (3) add 4 μL (40 units) of NEB TEV Protease Enzyme; (4) place sample in a tube rotator at 4°C, and (5) gently rotate sample overnight (≥16 hr). .. Day 10 – (1) Hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (2) repeat “ Buffer-Exchange (BE). .. Day 9 – (7) ” above to assess soluble protein concentration in supernatant b ; (3) add 1/9 sample volume (31.4 μL) of 10x IMAC-BB and 0.63 μL of 5.0 M imidazole (pH 7.8) to your sample to adjust chemical/salt concentrations to 16 mM HEPES, 420 mM NaCl, 0.008% azide, 50 m Na phosphate, 10 mM imidazole, pH ∼8; (4) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (5) remove His-tagged proteins from sample using one His-Spin Protein Miniprep (Zymo Research) and collect His-Spin FT containing RoxP-tagless; (6) perform BE into HSB using 2.0 mL Zeba Desalting Column (Thermo Scientific); (7) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (8) measure sample A 280 (DeNovix DS-11 FX+) to calculate the RoxP-tagless (GAT..ALN) concentration (ɛ280 13075 M -1 cm -1 , MW 14888.59 g/mol); (9) store RoxP protein sample at 4°C; and (10) verify BE and tag removal samples by SDS PAGE. a Reaction volume was scaled up as dictated by RoxP-tagless needs of planned experiments. b Only an estimation, as cleaved N-terminal tag is less soluble (i.e., may be in the post-centrifugation pellet) and a small amount of NEB TEV protease is present, though it is likely ≤5 μg per reaction.

    Protein Extraction:

    Article Title: Cutibacterium adaptation to life on humans provides a novel biomarker of C. acnes infections
    Article Snippet: Day 4 – (1) Incubate 10.0 mL of B-PER Complete Protein Extraction (stored at 4°C) in a ≥100 mL RT aluminum bead bath for ≥20 min to raise the temperature of this reagent to RT; (2) Incubate 25.0 mL of NiNTA-WB in an ice-bath for ≥30 min to prechill it to 4°C; (3) spin down 70 mL of ZYM-5052-Carb autoinduction culture (Eppendorf 5920 R, rotor FA-6x50; 10000 x g, 10 min, 4°C); (4) mass the cell pellet a ; (5) prepare 25 mL of RT B-PER with protease inhibitors (BPER-PI) b ; (6) collect cells by centrifugation (Eppendorf 5920 R, rotor FA-6x50, 10000 xg, 10 min, 4°C); (7) freeze-thaw the cell pellet (-20°C for 30 min then RT until thawed) c ; (8) gently resuspend cell pellet in BPER-PI and incubate with gentle inversion at RT for 15 min; (9) centrifuge lysate (Eppendorf 5920 R, rotor FA-6x50; 16000 x g, 20 min, 4°C) and transfer supernatant to a new 50 mL conical; (10) add 10 mL of ice-cold NiNTA-WB to lysate supernatant (1:1 lysate:buffer volume; imidazole final concentration: 10 mM; non-EDTA protease inhibitor final concentration: 1x); (11) filter sample; (12) concentrate to 2.0-4.0 mL using a Vivaspin 20 mL MWCO 10 d ; (13) hard spin sample (Eppendorf 5425 R, rotor FA-2x24; 21300 x g, 10 min, 4°C); (14) filter sample; (15) purify 8His-TEV-RoxP using one His-Spin Protein Miniprep column (Zymo Research) according to manufacturer’s instructions; (16) use a 2.0 mL Zeba Desalting Column (Thermo Scientific) to BE the His-Spin elution into PBS; (17) measure sample A 280 (DeNovix DS-11 FX+) to calculate 8His-TEV-RoxP concentration (MDH..ALN, ɛ280 14565 M -1 cm -1 , MW 17228.04 g/mol) e and yield f ; (18) add 26.7 μL of 10x NEB TEV Protease Reaction Buffer; (19) add 4 μL (40 units) of NEB TEV Protease Enzyme; (20) place sample in a tube rotator at 4°C, and (21) gently rotate sample overnight (≥16 hr). .. Day 5 – (1) Add 1/9 sample volume (30.1 μL) of 10x IMAC-BB and 0.60 μL of 5.0 M imidazole (pH 7.8) to the sample to adjust chemical/salt concentrations to 16 mM HEPES, 420 mM NaCl, 0.008% azide, 50 m Na phosphate, 10 mM imidazole, pH ∼8; (2) hard-spin sample (Eppendorf 5425 R, rotor FA-24x2, 21300 xg, 10 min, 4°C); (3) remove His-tagged proteins from sample using one His-Spin Protein Miniprep and collect His-Spin FT containing RoxP-tagless; (4) perform BE into HSB using 2.0 mL Zeba Desalting Column; (5) measure sample A 280 (DeNovix DS-11 FX+) to calculate the RoxP-tagless concentration (GAT..ALN, ɛ280 13075 M -1 cm -1 , MW 14888.59 g/mol) i and yield j ; (6) store RoxP protein sample at 4°C; and (7) verify BE and tag removal samples by SDS PAGE k . a Average cell pellet yield from 70 mL of autoinduction culture grown overnight at 18°C was 1.96 g, which is 78% of the cell pellet yield expected based on the larger scale (500 mL), 5-day at 18°C autoinduction culture. b A 10.0 mL aliquot of RT B-PER Complete Protein Extraction Reagent was supplemented with protease inhibitors aprotinin (4.0 μg/mL), leupeptin (40.0 μM), Pepstatin A (4.0 μM), PMSF (2.0 mM), and EDTA pH 8.0 (2.0 mM). ..



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