The 59 amino acid (aa 137-195) long rat SAC-Par-4 was cloned in pET-29b vector (Novagen) and expressed in BL21 (DE3); purified through HisPurTM Cobalt resin (Thermo Scientific) as described earlier (Sahoo et al., 2014a (link)). The purified SAC-Par-4 was dialyzed and concentrated with a centrifugal filter device (Amicon 10 KDa cut-off), and quantified by Bradford (Sigma). Fractions were collected and analyzed on 18% SDS-PAGE.
Hispur cobalt resin
Manufactured by Thermo Fisher Scientific
Sourced in United States, Germany
HisPur Cobalt Resin is a chromatography resin designed for the purification of proteins with a histidine (His) tag. It utilizes immobilized cobalt ions to selectively bind and capture His-tagged proteins from complex mixtures. The resin provides efficient protein capture and elution, enabling the purification of target proteins for various analytical and preparative applications.
Automatically generated - may contain errors
Lab products found in correlation
Imidazole, by Merck Group (5 mentions)
Autoinduction medium, by Formedium (4 mentions)
Desthiobiotin, by Merck Group (4 mentions)
Laemmli buffer, by Bio-Rad (4 mentions)
Qubit protein assay kit, by Thermo Fisher Scientific (4 mentions)
Penicillin streptomycin, by Thermo Fisher Scientific (4 mentions)
Protease inhibitor, by Merck Group (4 mentions)
Amicon ultra 15 centrifugal filter unit, by Merck Group (3 mentions)
Hitrap q anion exchange column, by GE Healthcare (3 mentions)
Complete protease inhibitor, by Merck Group (3 mentions)
Glutathione sepharose 4b, by GE Healthcare (3 mentions)
Streptactin column, by GE Healthcare (3 mentions)
Superdex 200, by GE Healthcare (3 mentions)
Sds page gel, by Thermo Fisher Scientific (3 mentions)
Dnase, by Merck Group (3 mentions)
Amylose resin, by New England Biolabs (3 mentions)
Protein assay, by Bio-Rad (3 mentions)
Pierce bca protein assay kit, by Thermo Fisher Scientific (3 mentions)
T4 dna ligase, by Thermo Fisher Scientific (3 mentions)
Amicon ultra 15, by Merck Group (2 mentions)
158 protocols using hispur cobalt resin
1
Recombinant Rat SAC-Par-4 Purification
2
Optimized Purification of Membrane Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
All experiments were performed at 4°C unless stated otherwise. The standard purification was described in Ma et al. (2015 (link)) and the optimized purification was carried out as follow. Membrane proteins (100 mg) were solubilised in purification buffer for 1 h. The non-solubilised material was removed by centrifugation (100,000 g for 1 h). The supernatant was collected and incubated with 4 ml HisPurTM Cobalt resin (Thermo ScientificTM) for 1 h on a roller mixer. The resin was then packed into a column and washed with 10 column volumes (CV) of wash buffer. The washed resin was then resuspended in 1 CV of cleavage buffer and incubated for 2 h with the appropriate amount of HRV-3C protease (molar ratio target protein to protease 1: 5). Afterwards, the flowthrough containing the protein of interest was collected and the resin was washed with 1 CV of cleavage buffer. Excess protease was removed from the protein solution by incubating it for 15 min with 4 ml of Ni-NTA resin slurry (Thermo ScientificTM) on a roller mixer. The cleaved protein was collected by transferring the suspension to an empty column and washing the resin with 1 CV of cleavage buffer. The protein was concentrated with a 30 kDa cut-off concentrator (Sartorius Stedim Biotech, 20 mL) for further experiments.
3
Affinity Purification of ACE2-coreSA Fusion
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The ACE2-coreSA fusion protein was extracted from the crude fractions using immobilized metal affinity chromatography. Initially, a 1:1 mixture of the crude protein and 10 mM imidazole (Acros Organics, New Jersey, NJ, USA) was made. The combination was applied to HisPurTM cobalt resin (Thermo Fisher Scientific), and incubated for 1 h under gentle shaking conditions at 4 °C. After loading the mixture onto the column, the flowthrough from the column was collected. The resin was washed at least five times with 10 mM imidazole. To elute the ACE2-coreSA fusion protein, 250 mM imidazole elution buffer was used. A protein concentrator (PES, MWCO = 50 K, Thermo Fisher Scientific) was used to concentrate the elution fractions. For the control experiments, ACE2 protein and coreSA protein with its own gene encoded in the pET-30a(+) vector were expressed, respectively, extracted and purified according to the aforementioned procedures.
4
Purification of His- and StrepII-tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human epsin1 and its mutants cloned with an N-terminal 6xHis tag and a C-terminal StrepII tag in pET15b have been described earlier (Holkar et al. 2015) (link). mEGFP was cloned with an N-terminal 6xHis tag in pET15b. Proteins were expressed in BL21(DE3) grown in autoinduction medium (Formedium, UK) at 18°C for 36 h. Bacterial cells were pelleted and stored at -40 °C. The frozen bacterial pellet was thawed and resuspended in 20 mM HEPES pH 7.4, 150 mM NaCl (HBS) with a protease inhibitor cocktail tablet (Roche) and lysed by probe sonication in an ice water bath. Lysates were spun at 18,000x g for 20 min. The supernatant was incubated with HisPur TM Cobalt Resin (Thermo Fisher Scientific) for 1 h at 4 °C and poured into a PD-10 column. The resin was washed with 100 ml of HBS and bound protein was eluted with HBS containing 250 mM imidazole. For StrepII tagged constructs, elution from the HisPur TM Cobalt Resin was applied to a 5 ml streptactin column (GE Healthcare) and washed with HBS. Proteins were eluted in HBS containing 1 mM DTT and 2.5 mM desthiobiotin (Sigma-Aldrich). Proteins were spun at 100,000x g to remove aggregates before use in assays. Protein concentration was estimated using UV absorbance at 280 nm based on the predicted molar extinction coefficients from the ProtParam tool in Expasy.
5
Purification of scFv Antibody Fragments
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Flow-cytometry-positive
scFvs were cloned into expression vector POE-myc. Details of cloning,
bacterial transformation, and induction are available inSupporting Information . Soluble scFv from periplasmic
extract was purified using HisPur Cobalt Resin (Life Technology, Grand
Island, NY). The periplasmic extract (40 mL) was first incubated with
1 mL resin for an hour with rotation, and then the resin was gravity-packed
in a column (1 × 1 cm). The resin column was washed with equilibration
buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole,
PH 7.4) until the A280 of flow through
reached a baseline. The scFv was subsequently eluted with 50 mM sodium
phosphate, 300 mM sodium chloride, 150 mM imidazole, pH 7.4. A few
fractions (1 mL each) of eluate were collected to ensure all protein
had been eluted, then those fractions with protein were pooled and
concentrated using an Amicon ultra-15 device (EMD Millipore, Billerica,
MA). The purified scFv was analyzed by SDS-PAGE and the protein concentration
was determined using the BCA protein assay (Pierce, Rockford, IL).
scFvs were cloned into expression vector POE-myc. Details of cloning,
bacterial transformation, and induction are available in
extract was purified using HisPur Cobalt Resin (Life Technology, Grand
Island, NY). The periplasmic extract (40 mL) was first incubated with
1 mL resin for an hour with rotation, and then the resin was gravity-packed
in a column (1 × 1 cm). The resin column was washed with equilibration
buffer (50 mM sodium phosphate, 300 mM sodium chloride, 10 mM imidazole,
PH 7.4) until the A280 of flow through
reached a baseline. The scFv was subsequently eluted with 50 mM sodium
phosphate, 300 mM sodium chloride, 150 mM imidazole, pH 7.4. A few
fractions (1 mL each) of eluate were collected to ensure all protein
had been eluted, then those fractions with protein were pooled and
concentrated using an Amicon ultra-15 device (EMD Millipore, Billerica,
MA). The purified scFv was analyzed by SDS-PAGE and the protein concentration
was determined using the BCA protein assay (Pierce, Rockford, IL).
6
Purification and Depletion of AQP4-Specific Ig
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
HEK293 cells (∼2*109) expressing C‐terminally His‐tagged AQP4 were lysed in 5ml of nondenaturing base lysis buffer (in mM: NaCl 300, Tris‐HCl [pH 7.4] 10, phosphate buffer [pH 7.4] 50, imidazol [pH 7.4] 10, 200 µl of ethylenedimainetetraacetic acid [EDTA]‐free protease inhibitor cocktail, and 5 µl of benzoase) with 1% decyl β‐D‐maltopyranoside for 40 minutes at room temperature. Lysate was centrifuged at 50,000g and cleared supernatant incubated with 500 µl of pre‐equilibrated HisPur Cobalt Resin (Life Technologies, Carlsbad, CA) for 1 hours. The resin was spun down and washed thoroughly with washing buffer I (as above, but containing only 0.01% decyl β‐D‐maltopyranoside) multiple times until absorption at 280nm became undetectable. Resin was resuspended in 40ml of 0.03% H2O2 and agitated at room temperature for 2 hours for oxidation of Co(II) to Co(III), spun down, and washed with wash buffer II (PBS and 100mM of EDTA) to strip away any unoxidized divalent ions.34 This AQP4 resin was used to deplete antigen‐specific Ig from samples. Control columns were prepared in the same way, however, using sham‐transfected HEK293 cells. For access to sufficient volumes, AQP4‐specific Ig depletion experiments were performed with NMO1 plasma.
7
Cloning and Purification of Bcd Homeodomain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A cDNA coding for amino acids 89–154 of the Bcd protein (including the homeodomain) as described in (Burz et al., 1998 (link)) using primers
5’-CAGCCAcatatgCTTTTCGATGAGCGAACG-3’
5’-GAGCCCggatccctaAGCGTAATCTGGAACATCGTATGGGTA CTGTTTCATACCCGGCGA-3’
to add a C-terminal HA epitope tag (underlined) and NdeI and BamHI sites (lowercase).
The PCR product was cloned into the pET-15b plasmid using NdeI and BamHI, which contains an N-terminal 6xHis tag and T7 promoter, to make plasmid pET-15B-BcdHD. Expression was induced in BL21 (DE3) pLysS E. coli cells using 2 mM IPTG. The protein was purified by affinity chromatography using HisPur Cobalt Resin (Fisher Scientific Cat # 89965) followed by ion exchange chromatography with SP Sepharose Fast Flow resin (GE Healthcare Cat #17-0729-01).
5’-CAGCCAcatatgCTTTTCGATGAGCGAACG-3’
5’-GAGCCCggatcccta
to add a C-terminal HA epitope tag (underlined) and NdeI and BamHI sites (lowercase).
The PCR product was cloned into the pET-15b plasmid using NdeI and BamHI, which contains an N-terminal 6xHis tag and T7 promoter, to make plasmid pET-15B-BcdHD. Expression was induced in BL21 (DE3) pLysS E. coli cells using 2 mM IPTG. The protein was purified by affinity chromatography using HisPur Cobalt Resin (Fisher Scientific Cat # 89965) followed by ion exchange chromatography with SP Sepharose Fast Flow resin (GE Healthcare Cat #17-0729-01).
8
Protein Purification and Screening Protocol
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Reagents were obtained from the following manufacturers: LB Broth, Ampicillin
Sodium Salt, HisPur Cobalt Resin (Fisher Scientific, Waltham, MA); IPTG (RPI
Corp., Mt. Prospect, IL); Complete EDTA-Free Protease Inhibitor Cocktail
Tablets, Lightcycler 480 II RT-PCR (Roche Applied Science, Indianapolis, IN);
SYPRO® Orange Protein Gel Stain 5000x Concentrate in DMSO (Life
Technologies, Grand Island, NY); 2-Mercaptoethanol, Electrophoresis ≥98%,
(Fisher BioReagents, Waltham, MA); DMSO (Sigma Aldrich, St. Louis, MO); Amicon
Ultra-15 Centrifugal Filter Units (EMD Millipore, Billerica, MA); MultiTron
Incubated Shaker (INFORS HT, Bottmingen, Switzerland); Ultrasonic Liquid
Processor (Misonix, Inc., Farmingdale, NY); ÄKTA purifier FPLC, Nanodrop
Spectrophotometer ND-1000 (GE Healthcare Life Sciences, Piscataway, NJ);
Envision 2104 multilabel reader (Perkin Elmer, Waltham, MA); Prestwick Chemical
Library® (Prestwick Chemical, Illkirch, France); NIH
Clinical Collection 1 and 2 (Evotec, San Francisco, CA); Custom Clinical
Collection (provided by Cliff Stephan at the GCC); National Cancer Institute
Diversity, Natural Products, Mechanistic, and Challenge Sets (NCI/NIH
Developmental Therapeutics Program, Bethesda, MD); BL21 Star™ (DE3),
Competent E. coli, PolarScreen™ ER-β Competitor
Assay, Green and PolarScreen™ ER-α Competitor Assay, Green (Life
Technologies, Carlsbad, CA).
Sodium Salt, HisPur Cobalt Resin (Fisher Scientific, Waltham, MA); IPTG (RPI
Corp., Mt. Prospect, IL); Complete EDTA-Free Protease Inhibitor Cocktail
Tablets, Lightcycler 480 II RT-PCR (Roche Applied Science, Indianapolis, IN);
SYPRO® Orange Protein Gel Stain 5000x Concentrate in DMSO (Life
Technologies, Grand Island, NY); 2-Mercaptoethanol, Electrophoresis ≥98%,
(Fisher BioReagents, Waltham, MA); DMSO (Sigma Aldrich, St. Louis, MO); Amicon
Ultra-15 Centrifugal Filter Units (EMD Millipore, Billerica, MA); MultiTron
Incubated Shaker (INFORS HT, Bottmingen, Switzerland); Ultrasonic Liquid
Processor (Misonix, Inc., Farmingdale, NY); ÄKTA purifier FPLC, Nanodrop
Spectrophotometer ND-1000 (GE Healthcare Life Sciences, Piscataway, NJ);
Envision 2104 multilabel reader (Perkin Elmer, Waltham, MA); Prestwick Chemical
Library® (Prestwick Chemical, Illkirch, France); NIH
Clinical Collection 1 and 2 (Evotec, San Francisco, CA); Custom Clinical
Collection (provided by Cliff Stephan at the GCC); National Cancer Institute
Diversity, Natural Products, Mechanistic, and Challenge Sets (NCI/NIH
Developmental Therapeutics Program, Bethesda, MD); BL21 Star™ (DE3),
Competent E. coli, PolarScreen™ ER-β Competitor
Assay, Green and PolarScreen™ ER-α Competitor Assay, Green (Life
Technologies, Carlsbad, CA).
9
Purification of Porcine CCL Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The established HEK 293 cells stably expressing porcine CCL proteins were harvested and resuspended in Gibco® FreeStyleTM 293 expression medium (Invitrogen), and incubated in the CELLSPIN system (INTERGRA bioscience, NH, USA) for three to four days. Supernatant was collected, centrifuged at 3000 rpm for 20 min, filtered through a 0.22 μm pore size membrane, and purified using HisPur cobalt resin (Invitrogen) with Econo-Column® chromatography (Bio-Rad laboratories, Hercules, CA, USA) following the manufacturer’s protocol. The eluted proteins were concentrated using Amicon® Ultra-15 (molecular weight cut-off 3 kDa, Millipore Corp., Bedford, MA, USA), mixed with cOmplete™ EDTA-free protease inhibitor cocktail (Roche Molecular Biochemicals, Laval, QC, Canada), and kept at −20 °C for further experiments.
10
Purification of KLF3 Recombinant Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Antigen KLF3 (14‐231AA) with C‐terminal His‐tag was extracted in the presence of 5 mol/L urea and 0.1% SDS from E coli BL21(DE3) plysS. Purification was made by HisPur Cobalt Resin (Invitrogen). The KLF3 rabbit polyclonal antibody was made in Biodragon Immunotech Company.
About PubCompare
Our mission is to provide scientists with the largest repository of trustworthy protocols and intelligent analytical tools, thereby offering them extensive information to design robust protocols aimed at minimizing the risk of failures.
We believe that the most crucial aspect is to grant scientists access to a wide range of reliable sources and new useful tools that surpass human capabilities.
However, we trust in allowing scientists to determine how to construct their own protocols based on this information, as they are the experts in their field.
Ready to get started?
Sign up for free.
Registration takes 20 seconds.
Available from any computer
No download required
Revolutionizing how scientists
search and build protocols!