Bio scale mini profinity imac cartridge

Manufactured by Bio-Rad

Sourced in United States, Germany

The Bio-Scale Mini Profinity IMAC Cartridges are lab equipment designed for the purification of recombinant proteins. They utilize immobilized metal affinity chromatography (IMAC) technology to capture and purify target proteins. The cartridges are pre-packed with a resin that binds to histidine-tagged proteins, allowing for efficient and reproducible protein purification.

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Lab products found in correlation

Ngc chromatography system, by Bio-Rad (4 mentions) Qubit protein assay kit, by Thermo Fisher Scientific (2 mentions) Pexp 5 ct topo ta expression vector, by Thermo Fisher Scientific (2 mentions) Ngc medium pressure chromatography system, by Bio-Rad (2 mentions) Enrich sec 70 size exclusion columns, by Bio-Rad (2 mentions) Tev protease, by New England Biolabs (1 mentions) Benzonase nuclease, by Merck Group (1 mentions) Bio scale mini bio gel p 6 desalting cartridge, by Bio-Rad (1 mentions) Biologic lp chromatographic system, by Bio-Rad (1 mentions) Qubit fluorimeter, by Thermo Fisher Scientific (1 mentions) Fraction collector, by Bio-Rad (1 mentions) Lp chromatography system, by Bio-Rad (1 mentions) Bio scale mini profinity gst cartridge, by Bio-Rad (1 mentions) Glutathione sepharose, by GE Healthcare (1 mentions) Q125 sonicator, by Qsonica (1 mentions) Hiload 16 60 superdex 200, by GE Healthcare (1 mentions) Hybond p, by GE Healthcare (1 mentions) Profinia protein purification system, by Bio-Rad (1 mentions) Ecl western blotting starter kit, by GE Healthcare (1 mentions) Chemidoc imaging system, by Bio-Rad (1 mentions)

17 protocols using bio scale mini profinity imac cartridge

Expression and Purification of WDR5 Truncation Mutant

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The protein analyte,
WDR5_ΔN,^{104 (link)} a truncation
WDR5 mutant lacking the residues 1–22, was expressed and purified
as previously described.^{87 (link),99 (link),104 (link)} The WDR5-containing supernatant underwent initial purification via
a metal-affinity column (5 mL, Bio-Scale Mini Profinity IMAC cartridge;
Bio-Rad, Hercules, CA). Then two enzymatic assays were performed on
the sample. Tobacco Etch Virus (TEV) protease (New England Biolabs)
removed the hexahistidine tag, and the benzonase nuclease (Sigma-Aldrich,
St. Louis, MO) digested DNA contaminants. Finally, the sample was
again passed through the metal-affinity column, and a 10 kDa molecular
weight concentrator (Millipore Sigma, St. Louis, MO) was used to concentrate
the final protein samples.

Mayse L.A., Imran A., Wang Y., Ahmad M., Oot R.A., Wilkens S, & Movileanu L. (2023). Evaluation of Nanopore Sensor Design Using Electrical and Optical Analyses. ACS Nano, 17(11), 10857-10871.

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Recombinant Production of Human Na+/K+-ATPase

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The recombinant extracellular portion of human Na+/K+-ATPase α1 subunit (M1-M2, M3-M4, M5-M6, M7-M8 and M9-M10) was expressed as a His-fusion protein using Escherichia coli, and purified by affinity chromatography. Briefly, the extracellular portion of the α1 subunit gene incorporating BamH I and Hind III sites was synthesized by Life Tech. Ltd. (Shanghai, China). The BamH I-Hind III fragment of the gene was subcloned into a pET-32b expression vector (Novagen) and transformed into E. coli BL21 (DE3) cells (Novagen). The BL21 transformants were cultivated in LB medium with ampicillin to an A600 nm of 0.8 at 37°C. Expression was induced by adding isopropyl β-D-thiogalactopyranoside (IPTG) to a final concentration of 10 mM, and incubated overnight at 20°C. The fusion protein containing a His-tag was isolated from the bacterial lysates by Ni²⁺-chelation affinity chromatography using a Bio-Scale^™ Mini Profinity^™ IMAC Cartridge (Bio-Rad). Finally, the recombinant protein was desalted using a Bio-Scale^™ Mini Bio-Gel P-6 Desalting Cartridge (Bio-Rad). The recombinant extracellular portions of the α2 and α3 subunits of human Na+/K+-ATPase were also expressed and purified using similar methods.

Liu M., Feng L.X., Sun P., Liu W., Wu W.Y., Jiang B.H., Yang M., Hu L.H., Guo D.A, & Liu X. (2016). A Novel Bufalin Derivative Exhibited Stronger Apoptosis-Inducing Effect than Bufalin in A549 Lung Cancer Cells and Lower Acute Toxicity in Mice. PLoS ONE, 11(7), e0159789.

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Recombinant Enzyme Production in E. coli

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The target sequence was adapted for obtaining recombinant enzyme in Escherichia coli cells and synthesized in ZAO Evrogen (Russia). The resulting sequence was cloned into the pET-23a (Novagen, USA) vector using NdeI and XhoI endonucleases to yield the target recombinant protein with His-tagged C-terminus. The resulting construction was transformed into Escherichia coli host strain BL21(DE3)pLysS (Novagen, USA). The heterologous expression of recombinant protein was performed in the LB/M9 mixed medium (1:1, by volume) supplemented with antibiotics at 37 °C until the cell culture reached an OD₆₀₀ of 0.6–0.8. The expression of the recombinant gene in E. coli cells was induced by adding 0.5 mM isopropyl-β-D-1-thiogalactopyranoside to the medium and the 5-aminolevulinic acid (100 mg/L), which facilitates the heme formation. The His-tagged recombinant protein was purified by immobilized metal affinity chromatography (IMAC) using Bio-Scale Mini Profinity IMAC cartridge and BioLogic LP chromatographic system (Bio-Rad, USA). The relative purity of the recombinant protein was measured by SDS-PAGE and staining of the gel with Coomassie brilliant blue R-250. Protein concentration was estimated as described before [48 (link)].

Toporkova Y.Y., Smirnova E.O., Lantsova N.V., Mukhtarova L.S, & Grechkin A.N. (2021). Detection of the First Epoxyalcohol Synthase/Allene Oxide Synthase (CYP74 Clan) in the Lancelet (Branchiostoma belcheri, Chordata). International Journal of Molecular Sciences, 22(9), 4737.

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Purification of Fn3 Protein from BL21(DE3)

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The biomass of BL21 (DE3) strain containing the pET16b:fn3 plasmid was resuspended in lysis buffer (50 mM NaH₂PO₄, 5 mM Tris-HCl, 300 mM NaCl, 10 mM imidazole, 1 mM PMSF, 5 mM DTT, pH 8.0) containing lysozyme (1 mg/ml), 0.5% Triton X-100 and 20 mM 2-mercaptoethanol and was sonicated. Insoluble particles were removed by centrifugation (7500 g, 30 min, 4 °C). The resulting lysate was filtered using a 0.22 μm pore size Millex-GP.
Protein isolation and purification were performed using a BioLogic LP chromatography system equipped with a fraction collector (Bio-Rad, USA). The clarified lysate was loaded onto a Bio-Scale™ Mini Profinity™ IMAC Cartridge (5 ml, Bio-Rad, USA), then equilibrated and washed with the lysis buffer, followed by another washing with the buffer (50 mM NaH₂PO₄, 5 mM Tris-HCl, 300 mM NaCl, 50 mM imidazole, 1 mM PMSF, 5 mM DTT, pH 8.0). The bound protein was eluted from the columns with buffer containing 300 mM imidazole. For further purification of the protein, dialysis was performed in PBS buffer containing 10% glycerol and 1 mM PMSF. The concentration of the isolated protein was measured by Qubit fluorimeter (Invitrogen, USA). The average yield of purified protein was approximately 20 mg per liter of E. coli culture. The purified protein was stored at −80 °C.

Dyakov I.N., Mavletova D.A., Chernyshova I.N., Snegireva N.A., Gavrilova M.V., Bushkova K.K., Dyachkova M.S., Alekseeva M.G, & Danilenko V.N. (2020). FN3 protein fragment containing two type III fibronectin domains from B. longum GT15 binds to human tumor necrosis factor alpha in vitro. Anaerobe, 65, 102247.

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METTL3-WTAP Protein Interaction Assay

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The human METTL3 gene was subcloned into pGEX-5X-2 expression plasmid with GST-tag and the human WTAP gene was subcloned into pProEX-HTb expression plasmid with His tag. Recombinant GST-METTL3 and His-WTAP proteins were induced to express in E. coli strain BL21(DE3) and purified by FPLC using Bio-Scale Mini Profinity GST cartridge (Biorad) and Bio-Scale Mini Profinity IMAC cartridge (Biorad) as described in the commercial instruction manual. The quality of proteins was tested by western blot and coomassie brilliant blue staining. His-WTAP proteins were incubated with glutathione sepharose (GE Healthcare) to be pre-cleared in NETN buffer (100 mM NaCl, 1 mM EDTA, 20 mM Tris-HCl pH 7.4, 0.5% NP-40). Then pre-cleared His-WTAP was mixed with GST or GST-METTL3 protein and incubated overnight at 4 °C with equal amount of glutathione sepharose beads. After washing the beads five times with NETN buffer, proteins bound to the beads were analyzed by 8% SDS-PAGE followed by immunoblotting with HRP-conjugated anti-His and anti-GST antibodies (Abcam).

Ping X.L., Sun B.F., Wang L., Xiao W., Yang X., Wang W.J., Adhikari S., Shi Y., Lv Y., Chen Y.S., Zhao X., Li A., Yang Y., Dahal U., Lou X.M., Liu X., Huang J., Yuan W.P., Zhu X.F., Cheng T., Zhao Y.L., Wang X., Danielsen J.M., Liu F, & Yang Y.G. (2014). Mammalian WTAP is a regulatory subunit of the RNA N6-methyladenosine methyltransferase. Cell Research, 24(2), 177-189.

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Purification of Polyhistidine-Tagged Proteins

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Bacterial cells were centrifuged
at 6000 rpm for 30 min at 4 °C and resuspended in 30 mL of cold
extraction buffer (50 mM Tris pH 8.00, 300 mM NaCl, 30 mM imidazole).
Phenylmethylsulfonyl fluoride (PMSF) was added to the cell suspension
to a final concentration of 1 mM. Cells were lysed via sonication
(Q125 Sonicator, QSonica). The cell lysate was centrifuged for 30
min at 30,000g at 4 °C to pellet the insoluble
fraction. Next, the polypeptide was purified using immobilized metal
ion affinity chromatography (IMAC). The sample was injected in an
IMAC column (Bio-Scale Mini Profinity IMAC cartridge, Bio-Rad Laboratories)
and washed with extraction buffer containing 2 M NaCl to remove DNA
contamination. The polypeptide was eluted with a linear gradient from
extraction buffer to elution buffer (50 mM Tris pH 8.00, 300 mM NaCl,
300 mM imidazole). The purity of the polypeptides was assessed by
sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Alvisi N., Zheng C., Lokker M., Boekestein V., de Haas R., Albada B, & de Vries R. (2022). Design of Polypeptides Self-Assembling into Antifouling Coatings: Exploiting Multivalency. Biomacromolecules, 23(9), 3507-3516.

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Production and Purification of DARPin Against Lamin A

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A DARPin against human lamin A, namely La_03 (Zwerger et al., 2015) , was modified to accommodate a cysteine at the N-terminus of the protein, cloned into pQE30. The original La_03 and the N-terminal Cys-DARPin proteins were produced in E. coli BL21(DE3). Transformation, growth and protein production in E. coli were carried out at 37 C. E. coli pellets were resuspended in 50 mM Tris-HCl pH7.4, 500 mM NaCl, 20 mM imidazole, 10% glycerol, and sonicated for 2 min with 20 sec intervals. The homogenate was centrifuged for 1 h at 16000 rpm, and the resulting supernatant was filtered through a 0.22 mm filter and loaded onto an affinity chromatography column (Bio-Scale Mini Profinity IMAC cartridge, BIO-RAD). DARPins were eluted with 50 mM Tris-HCl pH7.4, 500mM NaCl, 250 mM imidazole, 10% glycerol. Fractions containing DARPins were concentrated, loaded onto a size exclusion chromatography column (HiLoad 16/60 Superdex 200, GE Healthcare), and eluted with 20 mM Tris-HCl pH 7.4, 150 mM NaCl, 0.05% Tween-20. Finally, DARPins were concentrated and buffer-exchanged into a suitable conjugation buffer (PBS pH7.2, 1 mM EDTA).

Dahan I., Sorrentino S., Boujemaa-Paterski R, & Medalia O. (2018). Tiopronin-Protected Gold Nanoparticles as a Potential Marker for Cryo-EM and Tomography. Structure (London, England : 1993), 26(10).

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Quantification of GFP and Tsf-GFP in Floc Samples

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A floc sample (5 μL) was subjected to SDS-PAGE. For western blotting, proteins were transferred from the gel to a membrane sheet of Hybond P (GE Healthcare Ltd., UK) using the semi-dry transfer method. Hybridization was conducted using an anti-GFP primary antibody (Medical & Biological Laboratories Co., Japan) and an ECL Western Blotting Starter Kit (GE Healthcare Ltd.) according to the manufacturers' protocols.
Hybridization signals were detected using a ChemiDoc imaging system (Bio-Rad Laboratories Inc., Hercules, CA).
The protein bands were analyzed by densitometry (Image J software; NIH, Bethesda, MD) as an index of target protein expression. The amounts of GFP and Tsf-GFP were determined using purified GFP and Tsf-GFP as standards. Purification of GFP and Tsf-GFP was conducted using a Bio-scale Mini Profinity IMAC cartridge and Profinia protein purification system (Bio-Rad Laboratories Inc.) after ultrasonication of E. coli cells expressing each protein. Finally, the percentages of GFP and Tsf-GFP were determined based on total floc protein determined by the BCA method.

Ojima Y., Nunogami S., Azuma M, & Taya M. (2018). Displaying a recombinant protein on flocs self-produced by Escherichia coli through fused expression with elongation factor Ts. Enzyme and microbial technology, 108.

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Recombinant SN2 Peptide Expression

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The SN2 peptide was expressed in E. coli BL21[DE3] as previously described (Herbel, Schäfer & Wink, 2015 (link)). Purification was achieved by affinity chromatography using the Äkta start system (GE Healthcare, Solingen, Germany) combined with Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad, Munich, Germany). The SN2 peptide was expressed as a protein fused to thioredoxin to mask the antimicrobial activity during expression in the bacterial host. After purification of the fusion protein, thioredoxin was removed by TEV protease digestion (Herbel, Schäfer & Wink, 2015 (link)).

Herbel V, & Wink M. (2016). Mode of action and membrane specificity of the antimicrobial peptide snakin-2. PeerJ, 4, e1987.

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Recombinant Phage-Borne Endolysin Production

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The recombinant phage-borne endolysin was prepared according to the method described previously by Maciejewska et al.^{34 (link)}. Briefly, the coding sequence for Klebsiella phage KP27 endopeptidase was cloned into the commercially available pEXP-5-CT/TOPO® TA expression vector (Invitrogen, Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer recommendations, and BL21 (DE3) pLysS (Agilent Technologies, Santa Clara, CA, USA) was transformed with the expression construct. The expression was induced by the addition of isopropyl-β-D-thiogalactopyranoside (IPTG, final concentration of 0.5 mM), and bacteria further culture additional 18 h at 20 °C. The recombinant protein was purified from the filtered supernatant by affinity chromatography using NGC Medium Pressure Chromatography Systems (Bio-Rad, Hercules, CA, USA) combined with 5-ml nickel columns: Bio-Scale Mini Profinity IMAC Cartridges (Bio-Rad, Hercules, CA, USA) and dialyzed against PBS buffer. The concentration of purified recombinant enzyme was then determined fluorimetrically (Qubit® Protein Assay Kit, Molecular Probes, Thermo Fischer Scientific, Waltham, MA, USA).

Gałczyńska K., Ciepluch K., Madej Ł., Kurdziel K., Maciejewska B., Drulis-Kawa Z., Węgierek-Ciuk A., Lankoff A, & Arabski M. (2019). Selective cytotoxicity and antifungal properties of copper(II) and cobalt(II) complexes with imidazole-4-acetate anion or 1-allylimidazole. Scientific Reports, 9, 9777.

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