Purification was performed as described (8 (link)). Briefly, cells were thawed, disrupted with a microfluidizer M-110L (Microfluidics, Newton, MA), and centrifuged for 45 min at 45,000 g and 4°C to remove cell fragments. Supernatants were applied to a 25 mL nickel-nitrilotriacetic acid superflow column (Qiagen, Hilden, Germany) and eluted with buffers containing 200 mM imidazole. Fractions containing wild-type or mutant Gαi1 were screened via SDS-PAGE, pooled, concentrated to 5 mL using a 10,000 MWCO concentrator (Amicon Ultra-15, Merck), and applied to an illustra HiLoad 26/600 Superdex 200 pg column (GE Healthcare Life Sciences, Freiburg, Germany). Peak fractions were collected, concentrated to ca. 20 mg/mL, and concentrations were determined using Bradford reagent as triplicates. Wild-type or mutant protein was aliquoted, flash-frozen in liquid nitrogen, and stored at –80°C until utilization.
Nickel nitrilotriacetic acid superflow column
Manufactured by Qiagen
Sourced in Germany
The Nickel-nitrilotriacetic acid superflow column is a laboratory equipment used for protein purification. It is designed to efficiently capture and purify proteins with a histidine-tag. The column is pre-packed with a high-performance agarose resin that binds to the histidine-tag, allowing the target protein to be isolated from complex samples.
Automatically generated - may contain errors
Lab products found in correlation
Hiload 26 600 superdex 200 pg column, by GE Healthcare (1 mentions)
Amicon ultra 15, by Merck Group (1 mentions)
Plasmid isolation kit, by Qiagen (1 mentions)
Genomic dna extraction kit, by Promega (1 mentions)
Pet28a expression vector, by Qiagen (1 mentions)
Ex taq dna polymerase, by Promega (1 mentions)
T4 dna ligase, by New England Biolabs (1 mentions)
Pgem t easy vector, by Promega (1 mentions)
Oligonucleotide primers, by Bioneer (1 mentions)
2 protocols using nickel nitrilotriacetic acid superflow column
1
Purification of Recombinant Gαi1 Proteins
2
Molecular Cloning and Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Ex-Taq DNA polymerase, a genomic DNA extraction kit, pGEM-T easy vector, and reagents for polymerase chain reaction were purchased from Promega (Madison, WI, USA). T4 DNA ligase and restriction enzymes were purchased from New England Biolabs (Ipswich, MA, USA). The pET28a expression vector, plasmid isolation kit, and nickel-nitrilotriacetic acid superflow column for His-tag protein purification were obtained from Qiagen (Hilden, Germany)20 (link). Oligonucleotide primers were obtained from Bioneer (Daejeon, Republic of Korea)29 (link). Electrophoresis reagents were provided by Bio-Rad (Hercules, CA, USA), and all chemicals used in assays were purchased from Sigma-Aldrich (St. Louis, MO, USA).
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!