His-tagged recombinant LDHA and LDHB were expressed in E. coli and were purified by NTA nickel column (Qiagen, Hilden, Germany). The proteins were subjected to SDS-PAGE followed by Coomassie brilliant blue staining. The recombinant proteins were quantified based on the intensity of bands using the protein marker with known quantity (ProteinRuler II, TransGen Biotech, Beijing, China) as the reference. Next, the cell lysates were subjected to SDS-PAGE with quantified His-LDHA and LDHB and follow by WB using specific antibodies against LDHA and LDHB, respectively. Then the relative amount of endogenous LDHA and LDHB were quantified by comparing the intensity between His-tagged LDHA/B and endogenous LDHA/B based on the WB result using Image J. The relative expression levels of endogenous LDHA and LDHB in cell lines were then calculated according to the above results.
Nickel nta column
Manufactured by Qiagen
Sourced in Germany
The Nickel-NTA column is a laboratory tool used for the purification and isolation of proteins. Its core function is to facilitate the binding and elution of histidine-tagged proteins, allowing for their efficient separation and recovery from complex biological samples.
Automatically generated - may contain errors
6 protocols using nickel nta column
1
Quantification of Endogenous LDHA and LDHB
2
Plk1 Kinase Assay for Phosphorylation
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Protein expression was induced in BL21 bacteria by 0.5 mM isopropyl β-d -1-thiogalactopyranoside treatment at 16 °C. His-tagged proteins were purified using NTA Nickel column (Qiagen) and GST-tagged proteins were purified by GSH-conjugated agarose beads (GE). The purified proteins were concentrated. For kinase assay, the reaction was performed in 20 μl of 1× kinase buffer (50 mM NaCl, 2 mM EGTA, 25 mM HEPES, pH 7.2, 5 mM MgSO4, and 1 mM DTT) containing 100 ng of human recombinant Plk1, 2 μg of his-tagged protein and 50 μM ATP. For 32P assay, additional 5 μCi γ-32P ATP was added into the kinase reaction mixture, followed by incubation for 30 min at 30 °C. The reactions were stopped with 5× loading buffer and separated by 10% SDS-PAGE. For LC-MS-MS, the gel was stained with Coomassie Bright Blue, and the proteins in the gel were cut out for phosphorylation sites analysis by LC-MS-MS. For 32P assay, the protein in gel was transferred to the nitro-cellulose membrane, followed by exposure to X-ray film.
3
Incorporation of Fluorine-Labeled Tryptophans in R67 DHFR
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Fluorine‐labeled tryptophans were incorporated as described by Crowley et al.31 To facilitate protein purification, a his‐tagged construct of R67 DHFR cloned into a pRSETb vector, which has an additional 30 residues added to the disordered N‐terminus compared to the normal length R67 DHFR,21 was used. A W45F mutant was prepared as described.17 Cells were grown in rich media at 37°C to an O.D. 600 of 0.6. The cells were harvested by centrifugation and the pellet was resuspended in an equal volume of pre‐warmed minimal medium (7.5 mM [NH4]2SO4, 50 mM Na2HPO4 and 50 mM KH2PO4 pH 7.0). 4‐, 5‐, 6‐ or 7‐Fluoroindoles were dissolved in dimethyl sulfoxide and added to the media (final concentration of 60 mg/L). After 30 minutes of acclimatization, protein expression was induced with the addition of 1 mM IPTG for 10 h. Protein was purified on a nickel‐NTA column (Qiagen).8 Purified protein was dialyzed against deionized H2O, lyophilized, and stored at 4°C. α‐Chymotrypsin‐truncated R67 DHFR was prepared as described.18 , 19 Protein concentrations were determined by measuring the absorbance at 280 nm using extinction coefficients (1.15, 1.38, 1.35, and 1.19 ml/mg for 4F, 5F, 6F, and 7F R67 DHFR, respectively) determined with a bicinchoninic acid assay (Pierce).
4
Expression and Purification of Dok1 PTB Domain
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human Dok1 PTB domain (Q154 – G256; Swiss-Prot Q99704, number as Q1-G103) was sub-cloned into the pET14b vector with an N-terminal six His-tag. Escherichia coli Rosetta cells were transformed with the expression plasmid and grown at 37 °C either in LB or M9 medium containing [15N] ammonium chloride (Cambridge Isotope Laboratories). Expression of recombinant protein was induced by adding 1 mM IPTG to cells (OD600 0.6–0.7) followed by incubation for 6–12 h at 16 °C. Cells were centrifuged and re-suspended in Buffer A (20 mM Tris-HCl buffer, pH 8.0) followed by affinity purification on a Nickel-NTA column (Qiagen). His-tagged Dok1 PTB domain was eluted in Buffer A containing 500 mM imidazole and dialyzed against Buffer B (50 mM sodium phosphate buffer (pH 6.0) containing 100 mM NaCl and 2 mM DTT). Protein samples were further purified by gel filtration in Buffer B on a Hiload Superdex 75 16/26 GL preparative column that was connected to an AKTA FPLC UPC-900 system (GE Healthcare UK Ltd., England). Proteins were eluted at a flow rate of 0.5 ml/min and monitored by absorbance at λ = 276 nm.
5
Cloning and Purification of RimR2 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
A 2757-bp DNA fragment harboring the rimR2 coding sequence was amplified by PCR using primers PrimR2-F2/R3 (Additional file 15 : Table S2) and S. rimosus M527 genomic DNA as a template. The rimR2 gene was inserted into the EcoR V and BamH I sites of pET32a to generate the plasmid pET32a-rimR2. Then, plasmid pET32a-rimR2 was expressed in E. coli BL21 (DE3). RimR2 protein was purified using nickel-NTA column (Qiagen) and eluted using imidazole. The inducible expression and purification of were performed according to standard manipulation method described by Sambrook and Russel [52 ].
6
Purification of Recombinant SOX9 Protein
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Typically, 1–2 L cultures were grown at 25°C in LB media. Upon induction with 1 mM isopropylthiogalactoside at an A600nm of 0.8, cultures were grown five hours further. As all expressed proteins were insoluble, the cell pellet was dissolved in a denaturing solution of TP300 (20 mM Tris-HCl + 50 mM sodium phosphate + 300 mM NaCl, pH 7.5) supplemented with 6 M urea and lysed further with a French press. The solubilized SOX9 protein was bound to a 10 mL nickel-NTA column (Qiagen) in the same buffer, successively washed with TP300 + 4 M urea, TP300 + 2 M urea, TP300 + 1 M urea and TP300 + 15 mM imidazole and eluted with ice cold TP300 + 20 mM EDTA + 1 mM phenylmethylsulfonylfluoride. After a brief concentration step, the protein was purified further by size exclusion chromatography using a Sephacryl S100 16/60 HiLoad column (GE Biosciences) equilibrated with 10 mM sodium phosphate buffer + 100 mM NaCl, pH 6.0. Protein concentrations were determined from their extinction coefficient at 280 nm as calculated by ProtParam [20 ].
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!