Exon 1 of the HTT huntingtin (NCBI Gene ID 3064) was expressed as a fusion protein with (i) TRX (thioredoxin) only (MurTRX); (ii) 16Q (Mur16); and (iii) 46Q (Mur46). MurTRX, Mur16, and Mur46 were produced and purified by HIS-tag and size exclusion chromatography (SEC). Colonies were picked and 5 mL cultures were grown. Cells were induced at Optical Density (OD) 0.7–0.8 for four hours at 37 °C. 1 mL of the post-induction culture was spun down and the pellet resuspended in lysis buffer. This was spun down at 10,000 RPM for 10 min and the soluble fraction loaded on a 4–20% polyacrylamide gel (NuSep). An anti-His western blot was performed to detect protein expression. An ELISA assay was performed to confirm the expression and detection of the proteins. MurTRX, Mur16 and Mur46 are recognized by anti-His antibodies. Mur16 and Mur46 are recognized by the mHTT antibody 3B5H10 (Cat MABN821, Merck, Germany). Proteins were loaded onto IMAC resin (ThermoFisher Scientific HisPur) and eluted in 200 mM imidazole, purified by gel filtration FPLC (HiLoad superdex-200, 26/60; GE Life sciences) and concentrated using 3 kDa cut off Vivaspin 20 PES centrifugal concentrators (Sartorius AG). Proteins were biotinylated using a 1:0.5 molar ratio of EZ-link™ Sulfo-NHS-LC-LC-Biotin (ThermoFisher Scientific) and thoroughly dialysed against PBS prior to Biacore coupling.
Hispur
Manufactured by Thermo Fisher Scientific
HisPur is a nickel-charged resin for the purification of histidine-tagged recombinant proteins. It is designed to efficiently capture and purify His-tagged proteins from a variety of sample types.
Automatically generated - may contain errors
Lab products found in correlation
Thrombin cleancleave kit, by Merck Group (2 mentions)
Amylose, by New England Biolabs (2 mentions)
Ez link sulfo nhs lc lc biotin, by Thermo Fisher Scientific (1 mentions)
Hiload superdex 200, by GE Healthcare (1 mentions)
Superdex 200 10 300 gl, by Cytiva (1 mentions)
Akta pure fplc system, by Cytiva (1 mentions)
Superdex g 75, by GE Healthcare (1 mentions)
Gst trap column, by GE Healthcare (1 mentions)
B per, by Thermo Fisher Scientific (1 mentions)
Complete edta free, by Merck Group (1 mentions)
Dneasy blood and tissue kit, by Qiagen (1 mentions)
Ni nta column, by Thermo Fisher Scientific (1 mentions)
Superdex 200 16 60 column, by GE Healthcare (1 mentions)
Phusion polymerase, by Thermo Fisher Scientific (1 mentions)
10 protocols using hispur
1
Expression and Purification of HTT Fusion Proteins
2
Recombinant Expression and Purification of MCUb
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The nucleotide sequence encoding the NTD of H. sapiens MCUb (GenBank: NP_060388.2) was cloned into a pET-28a vector using the NheI and XhoI restriction sites. Transformed BL21(DE3) codon plus E. coli cells were grown in Luria-Bertani (LB) medium containing kanamycin (60 μg mL−1) at 37°C until the optical density (OD) at 600 nm reached ∼0.6–0.8. Subsequently, 0.35 mM of isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to induce 6×His-MCUb58-159 expression over 16 h at 24.5°C. Harvested cells were lysed by sonication in 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM DTT. Purification was performed as described in the nickel-nitrilotriacetic acid (Ni2+-NTA) agarose beads manufacturer protocol (HisPur, Thermo Fisher). The 6×His tags were removed by overnight incubation with ∼1 Unit of bovine thrombin (EMD millipore) per mg of protein. Size-exclusion chromatography (SEC) through a Superdex 200 10/300 GL (Cytiva), connected to an AKTA pure FPLC system (Cytiva) at 10°C, was performed as the final purification step in 20 mM Tris (pH 8.5), 150 mM NaCl, 1 mM DTT. The protein concentration of MCUb58-159 was estimated using an extinction coefficient (280 nm) of 0.4280 (mg/mL)−1 cm−1.
3
Purification of His6-SMT3 Fusion Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
MPL mutants were expressed as His6-SMT3 fusions in BL21 (DE3) E.coli, purified by Ni-NTA (Thermo Scientific HisPur) affinity chromatography, cleaved by ULP1-His6 and separated by reverse Ni-NTA, followed by gel filtration chromatography on a Superdex G75 preparative column (GE Healthcare). MemAb trastuzumab was expressed and purified as previously described8 (link).
4
Bacterial Protein Expression and Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Expression strains were grown in 1 L LB Amp100 Cm10 at 37°C to an OD600 of 0.5, induced with 1 mM IPTG, shifted to 30°C, grown an additional 3 hours, and harvested at 8,000 x g. Proteins were purified at 4°C by the batch method over 1 mL Ni-NTA resin according to the manufacturer’s instructions (HisPur, Thermo Scientific). All buffers were 50 mM NaPO4, pH 7.4, 100 mM NaCl, with varied imidazole (10 mM lysis buffer, 20 mM wash buffer, 250 mM elution buffer). The eluted protein was dialyzed against 50 mM NaPO4, pH 7.4, 100 mM NaCl and either stored at 4°C or adjusted to 5% glycerol and stored at −80°C.
5
Kinase Activity Assay for SMG1 and p53
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
His-tagged, wild type (WT) or kinase dead (DA) SMG1 ectopically expressed 293T cells was immunoprecipitated using cobalt beads (HisPur™, ThermoFischer) in a buffer containing 300 mM NaCl, 50 mM Na3PO4 (pH 8.0), 0.02% Tween, 0.25% NP40, and protease inhibitor cocktail (EDTA-free, Pierce). A recombinant GST-p53N fusion protein containing a N-terminal segment of p53 with the Ser15-phosphorylation site was expressed in E. coli and purified using FPLC with a GSTTrap column (GE Healthcare). In vitro kinase reaction was performed by incubating His-SMG1 (WT) or His-SMG1 (DA) with the GST-p53 substrate in the presence or absence of CC (10 μM) or caffeine (10 mM) at 30°C for 2 hours in a kinase buffer (25 mM Tris HCl, pH 7.5, 10 mM β-Glycerophosphate, 0.2 mM Na3VO4, 10 mM MgCl2, 0.05 mM DTT). Reactions were terminated by the addition of SDS sample buffer. S15-phosphorylation of the GST-p53N substrate was detected by western blot using a phosphor-specific antibody (Cell Signaling Technology, 9284).
6
Purification of Vaginolysin Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
DNA was extracted from G. vaginalis strain AMD using the DNeasy Blood and Tissue kit (Qiagen). The vly gene was amplified using primers VaginolysinFWD (5’-GGAAGGGATCCGATTCTTCTGCAAAGCCTTCTGC-3’ ) and VaginolysinREV (5’-GGAAGCTCGAGTCAGTCATTCTTTACAGTTTCAGCAAC-3’ ) as previously described [28 (link)]. Purified PCR product was restricted with BamHI and XhoI and ligated to pET32. Plasmid from a colony that grew on LB agar containing 100 μg ampicillin / mL was confirmed by DNA sequencing and transformed into E. coli strain BL21(DE3) CodonPlus pRIPL (Agilent technologies). Cultures were grown in 1 L LB containing 100 μg amp / mL and 35 μg chloramphenicol / mL to exponential phase, induced with 1 mM IPTG for 2 hours, and the bacteria were collected by centrifugation. Bacteria were lysed in a French pressure cell in B-PER™ (ThermoFisher Scientific) containing protease inhibitors (EDTA-free cOmplete™, Sigma) and the lysate was cleared by centrifugation and filtration. The protein was purified by cobalt affinity chromatography (His-Pur™, Thermo-Fisher Scientific) according to manufacturer instructions, eluted in 0.25 M imidazole, and dialyzed against 1X phosphate buffered saline (PBS). The affinity tag was removed by thrombin digestion (Sigma-Aldrich, Thrombin CleanCleave Kit).
7
Generating Mutated TGM-D13 Constructs
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Constructs coding for H. polygyrus TGM-D3 and TβRII described previously were modified to introduce the desired substitution using site-directed mutagenesis with Phusion polymerase (ThermoFisher) as previously described (64 (link)). The resulting clones were sequenced over the entirety of their coding sequences to confirm the substitution. Constructs coding mutated forms of TGM D1-D3 (TGM D13) were generated by synthesis of coding sequences for TGM D13, identical to those described previously for TGM-1 D13, but with the desired substitution and then inserted into AscI- and ApaI-digested pSec-Tag2 as described previously (35 (link)). Desired constructs, which code for TGM D13 downstream of a signal peptide and with a C-terminal myc-tag and hexahistidine tag, were transfected into suspension cultured expi293 cells, and after 5 days, the protein was purified from the conditioned medium by capturing it on a NiNTA column (Thermo, His-Pur). The purified TGM D13 was pooled, deglycosylated with PNGase-F, concentrated, and further purified on Superdex 200 16/60 column (GE Lifesciences).
8
In Vitro Phosphorylation of Drosophila Plk4 and Ana2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterially expressed constructs of Drosophila Plk4 (aa 1–317) C-terminally tagged with FLAG-His6 and FL Drosophila Ana2 N-terminally tagged with glutathione S-transferase (GST-Ana2) were purified on HisPur (Thermo Fisher Scientific), amylose (NEB), or glutathione resin (NEB), respectively, according to the manufacturers’ instructions. In vitro phosphorylation assays were performed by incubating 5–10 μM Plk4, 20 μM Ana2, and 100 μM ATP for 1–2 h at 22°C in RXN1 buffer (40 mM Na Hepes, pH 7.3, 150 mM NaCl, 5 mM MgCl2, 1 mM DTT, and 10% [by volume] glycerol). Phosphorylated residues within proteins were identified by MS/MS (Table S1) of purified bacterially expressed proteins phosphorylated in vitro (described above) in the presence of nonradioactive ATP.
9
Thrombin Cleavage of His-Tagged Proteins
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Following Ni-NTA purification, FecB and FecB2 proteins were treated with the thrombin CleanCleave kit (Sigma) following manufacturer protocols to remove His-tags. Briefly, thrombin resin was added to the protein and incubated overnight at 4°C with stirring. The resin was then removed by centrifugation at 2,500 rpm for 5 minutes and the cleaved protein sample was incubated with Ni-NTA resin (HisPur, Thermo Scientific) for one hour at room temperature. The flow-through was collected and verified to contain cleaved FecB or FecB2 by SDS-PAGE and MALDI-TOF mass spectrometry.
10
Purification and Binding of Drosophila Ana2
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Bacterially expressed constructs of Drosophila Ana2 (aa 1–317 or 318–420) N-terminally tagged with His6 or MBP were purified on HisPur (Thermo Fisher Scientific) or amylose (NEB), respectively, according to the manufacturers’ instructions. MBP-Ana2 was not eluted from the resin for binding assays. Proteins were analyzed by SDS-PAGE, and equimolar amounts of MBP-Ana2 and His6-Ana2 were mixed together in buffer RXN1. Samples were incubated for 30 min at room temperature with shaking, followed by eight washes with buffer RXN1. Resin was resuspended in sample buffer and subsequently run on SDS-PAGE for analysis by Western blotting.
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!