Synthetic genes with codons optimized for E. coli expression were from Genscript. BL21(DE3) cells with pET 28 vectors were grown in a fermenter and cells were broken and initially processed as previously described55 (link). The proteins: full-length human p.BACH21–841 and p.L24P variant; murine p.Bach21–133 and murine p.Bach21–133 L24P all contained an N-terminal his-tag to facilitate purification (NB The sequence difference between human p.BACH21–133 and murine p.Bach21–133 is at one position, amino acid 8, which is Asp in human and Ala in murine). Human WT p.BACH21–841 was extracted from cell lysate with 100 mM sodium bicarbonate, pH 9.5 containing 2 M urea and the L24P variant with 8 M guanidine-HCl. WT proteins were expressed as a soluble protein but L24P variants were insoluble and extracted with 8M guanidine-HCl. Proteins were purified using a combination of Ni-chelate and size exclusion chromatographies using Ni-chelate Sepharose and Sephadex S200 (both from GE Healthcare). The L24P variants were folded by dialysis against 4 M urea and then stepped through lower concentrations until the urea was removed. DTT was present in all buffers to keep proteins reduced.
Sephadex s200
Manufactured by GE Healthcare
Sourced in United Kingdom, United States
Sephadex S200 is a gel filtration medium used for the separation and purification of biomolecules. It is composed of cross-linked dextran beads and is designed for size-exclusion chromatography applications. The product's core function is to separate and isolate proteins, nucleic acids, and other macromolecules based on their molecular size and shape.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using sephadex s200
1
Purification and Folding of BACH2 Variants
2
Protein Purification via Chromatography
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Chromatography columns HiTrap Heparin (1 and 5 mL), Sephadex S200 and the chromatography equipment AKTA-explorer were from GE Healthcare (Little Chalfont, UK). Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) reagents were from BioRad (Hercules, CA, USA). Unless otherwise stated, other chemicals were purchased from Sigma-Aldrich (St. Louis, MO, USA).
3
Purification and Folding of BACH2 Variants
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Synthetic genes with codons optimized for E. coli expression were from Genscript. BL21(DE3) cells with pET 28 vectors were grown in a fermenter and cells were broken and initially processed as previously described55 (link). The proteins: full-length human p.BACH21–841 and p.L24P variant; murine p.Bach21–133 and murine p.Bach21–133 L24P all contained an N-terminal his-tag to facilitate purification (NB The sequence difference between human p.BACH21–133 and murine p.Bach21–133 is at one position, amino acid 8, which is Asp in human and Ala in murine). Human WT p.BACH21–841 was extracted from cell lysate with 100 mM sodium bicarbonate, pH 9.5 containing 2 M urea and the L24P variant with 8 M guanidine-HCl. WT proteins were expressed as a soluble protein but L24P variants were insoluble and extracted with 8M guanidine-HCl. Proteins were purified using a combination of Ni-chelate and size exclusion chromatographies using Ni-chelate Sepharose and Sephadex S200 (both from GE Healthcare). The L24P variants were folded by dialysis against 4 M urea and then stepped through lower concentrations until the urea was removed. DTT was present in all buffers to keep proteins reduced.
4
Recombinant Human SR Protein Purification
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Human SR gene (Accession: NM_021947.1) was commercially purchased from Gene Copoeia, USA. The gene (1020 bp) was PCR amplified using gene specific primers and subcloned into pProEX HTa vector. The protein was expressed in E. coli rosetta cells. The protein was purified using Ni-NTA affinity chromatography in 50 mM Tris-HCl, pH 8.0 containing 25 µM PLP (with 300 mM imidazole as eluting agent). It was further purified using Sephadex S-200 (GE Healthcare, USA) in AKTA Prime (GE Healthcare, USA) with a flow rate of 1 ml/min of 20 mM Tris-HCl, 150 mM NaCl, and 15 μM PLP pH 8.0. The purified protein was identified by mass spectrometry and was resolved in SDS-PAGE to test.
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!