Full-length PfcFRS was purified according to the earlier published report2 (link). Full-length PvcFRS was also purified according to the same protocol. In brief, the genes encoding PvcFRS alpha subunit (PVX_081300) and beta subunit (PVX_090880) were cloned into E. coli pETM11 and pETM20 plasmids respectively. Both plasmids were co-transformed into E. coli strain B834 and were induced overnight for overexpression with 0.5 mM isopropyl-β-D-thiogalactoside (IPTG) at 16 ° C for 18 h. The E. coli cell lysate was first loaded onto a nickel–nitrilotriacetic (Ni–NTA) column (GE Healthcare) and the eluted fraction was further purified with Heparin chromatography (GE Healthcare) to a single band as indicated by SDS–polyacrylamide gel electrophoresis with Coomassie brilliant blue staining. The purified protein was found as a single peak with the elution volume consistent of a homogeneous PvcFRS hetero-tetramer on the Superdex 200 analytical gel filtration column (GE Healthcare). The purified PvcFRS was concentrated to 25 mg ml−1 and stored at −80 °C in 25 mM HEPES buffer, pH 7.5, 200 mM NaCl, 5 mM βME.
Heparin chromatography
Manufactured by GE Healthcare
Sourced in United States
Heparin chromatography is a separation technique used to purify and analyze heparin, a naturally occurring anticoagulant. It involves the use of a heparin-binding column to selectively capture and separate heparin from other components in a sample. The core function of heparin chromatography is to provide a reliable and efficient method for the isolation and characterization of heparin, which is widely used in various medical and pharmaceutical applications.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using heparin chromatography
1
Purification of Pvcfrs and Pfcfrs Proteins
2
Purification of Fluorescent-Tagged E. coli UvrA
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
The gene for E. coli UvrA, obtained from National Bioresource Project (NIG, Kyoto, Japan), was engineered onto a C-terminal mScarlet fluorescent protein separated via a flexible linker (20 (link)). Further C-terminal to mScarlet, we placed a His tag for purification and induced expression of this pET21a construct overnight at 18°C using isopropyl β-d -1-thiogalactopyranoside. Purification was performed using nickel affinity followed by heparin chromatography (GE Healthcare, Chicago, IL, USA). To avoid protein precipitation, we kept KCl concentrations >200 mM. Once purified, the UvrA concentration was determined using mScarlet’s extinction coefficient (M−1cm−1) at 569 nm and stored in 50% glycerol 50 mM Tris (pH 7.5), 500 mM KCl, 0.1 mM EDTA, and 10 mM DTT at −20°C (18 (link)).
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!