The catalytic domain of drosophila TET (dTET) was cloned in pET28a expression vector. The His-tagged protein was overexpressed in E. coli BL21 (DE3) CodonPlus RIL cells for 17 h at 16 °C. Cells were resuspended in lysis buffer (50 mM HEPES pH 7.5, 20 mM imidazole, 500 mM NaCl, 1 mM DTT, 10% glycerol, and supplemented with protease inhibitor 0.2 mM PMSF) and disrupted using Bandelin Sonoplus ultrasonic homogenizer. The cell lysates were cleared by centrifugation (Lynx 600 (Thermo), Fiberlite F21−8x50 y) at 38.300 g for 30 min at 4 °C and the supernatant was loaded onto an affinity column packed with Ni-NTA agarose beads (Genaxxon, Germany). The column was washed with wash buffer (50 mM HEPES pH 7.5, 20 mM imidazole, 500 mM NaCl, 1 mM DTT, 10% glycerol) then the recombinant protein was eluted with elution buffer (50 mM HEPES pH 7.5, 250 mM imidazole, 500 mM NaCl, 1 mM DTT, 10% glycerol). Purified protein was dialyzed against dialysis buffer I (50 mM HEPES pH 7.5, 1 mM DTT, 300 mM NaCl, and 10% glycerol) followed by dialysis buffer II (50 mM HEPES pH 7.5, 1 mM DTT, 300 mM NaCl, and 50% glycerol).
Fiberlite f21 8 x 50y
Manufactured by Thermo Fisher Scientific
Sourced in Germany
The Fiberlite™F21-8 x 50y is a high-performance centrifuge rotor designed for use in laboratories. It features a maximum speed of 21,000 RPM and a maximum RCF of 50,000 x g. The rotor has a capacity of 8 tubes and is compatible with various tube sizes.
Automatically generated - may contain errors
Lab products found in correlation
3 protocols using fiberlite f21 8 x 50y
1
Purification of Drosophila TET Catalytic Domain
2
Affinity Purification of His-Tagged Protein Complexes
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
Cell pellet was resuspended in buffer (25 mM Tris-HCl pH 8.0, 150 mM
NaCl, 5 mM Imidazole, EDTA free complete protease inhibitor (Roche), lysed by 2
cycles of freeze thaw in liquid nitrogen and the lysate was cleared by
centrifugation at 19,000 g in a Thermo Scientific™ Fiberlite™
F21-8 x 50y fixed-angle rotor for 90 min. The supernatant was mixed with Talon
resin (Clontech), equilibrated in buffer A (25 mM Tris-HCl pH 8.0, 150 mM NaCl,
5 mM Imidazole) and incubated for 1 hour at 4° C. The resin was washed
with 15 column volumes (CV), 10 CV of buffer HS (25 mM Tris-HCl pH 8.0, 1000 mM
NaCl, 5 mM Imidazole), and 15 CV of buffer A before eluting the bound protein
complex with 5 CV of buffer B (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 200 mM
Imidazole). Different samples were analysed by SDS-PAGE analysis and western
blot analysis using HRP conjugated anti-His antibody (Sigma-Aldrich) for TAF5NTD
and anti-TAF6 (primary, received from Laszlo Tora) followed by HRP conjugated
anti-mouse (secondary, Sigma-Aldrich) for TAF6.
NaCl, 5 mM Imidazole, EDTA free complete protease inhibitor (Roche), lysed by 2
cycles of freeze thaw in liquid nitrogen and the lysate was cleared by
centrifugation at 19,000 g in a Thermo Scientific™ Fiberlite™
F21-8 x 50y fixed-angle rotor for 90 min. The supernatant was mixed with Talon
resin (Clontech), equilibrated in buffer A (25 mM Tris-HCl pH 8.0, 150 mM NaCl,
5 mM Imidazole) and incubated for 1 hour at 4° C. The resin was washed
with 15 column volumes (CV), 10 CV of buffer HS (25 mM Tris-HCl pH 8.0, 1000 mM
NaCl, 5 mM Imidazole), and 15 CV of buffer A before eluting the bound protein
complex with 5 CV of buffer B (25 mM Tris-HCl pH 8.0, 150 mM NaCl, 200 mM
Imidazole). Different samples were analysed by SDS-PAGE analysis and western
blot analysis using HRP conjugated anti-His antibody (Sigma-Aldrich) for TAF5NTD
and anti-TAF6 (primary, received from Laszlo Tora) followed by HRP conjugated
anti-mouse (secondary, Sigma-Aldrich) for TAF6.
3
Affinity Purification of His-Tagged Protein Complexes
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!