Transformed with the expression vector Pet15b containing either SUMO-MTS-EmGFP, SUMO-MTS-SNAP-tag or SUMO-SNAP-tag E. coli cells were grown in lysogeny broth (LB) medium up to OD600∼1.0 and induced with 1 mM IPTG (Helicon, Moscow, Russia). The cells were harvested after 3 h, centrifuged at 5000 × g for 10 min, and the pellet was frozen at −80°C. The cell lysis was performed either by a microfluidizer or ultrasound (depending on the amount of cells), in the lysis buffer (300 mM NaCl 50 mM NaH2PO4 pH = 7.0). The lysate was centrifuged at 10000 × g for 1 h, and the supernatant was applied onto affinity resin Ni-NTA (QIAGEN, Düsseldorf, Germany) in a column. After washing the column with the lysis buffer, the protein was eluted with 200 mM imidazole dissolved in the lysis buffer. Imidazole was removed by dialysis against the cell buffer: 130 mM KCl, 10 mM NaCl, 2 mM CaCl2, 20 mM NaHCO3 pH = 7.2. After dialysis, the solution was filtered through a 0.22 μm syringe filter (Merck Millipore, Darmstadt, Germany) and stored at 4°C.
Isopropyl β d 1 thiogalactopyranoside (iptg)
Manufactured by Helicon
IPTG (Isopropyl β-D-1-thiogalactopyranoside) is a synthetic chemical compound commonly used as an inducer in bacterial expression systems. It functions by binding to and inactivating the lac repressor protein, allowing transcription of genes under the control of the lac promoter to occur.
Automatically generated - may contain errors
Lab products found in correlation
2 protocols using isopropyl β d 1 thiogalactopyranoside (iptg)
1
Purification of Fluorescently-Tagged Recombinant Proteins
2
Purification of SUMO-Tagged Proteins
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Transformed with the expression vector Pet15b containing either SUMO-MTS-EmGFP, SUMO-MTS-SNAP-tag or SUMO-SNAP-tag E. coli cells were grown in lysogeny broth (LB) medium up to OD600~1.0 and induced with 1mM IPTG (Helicon, Moscow, Russia). The cells were harvested after 3 h, centrifuged at 5000 g for 10 min, and the pellet was frozen at -80° C. The cell lysis was performed either by a microfluidizer or ultrasound (depending on the amount of cells), in the lysis buffer -300 mM NaCl 50 mM NaH2PO4 pH=7. The lysate was centrifuged at 10000 g for 1 h, and the supernatant was applied onto affinity resin Ni-NTA (Qiagen, Dusseldorf, Germany) in a column. After washing the column with the lysis buffer, the protein was eluted with 200 mM imidazole dissolved in the lysis buffer. Imidazole was removed by dialysis against the cell buffer: 130 mM KCl, 10 мМ NaCl, 2mM CaCl2, 20 mM NaHCO3 pH=7.2. After the dialysis, the solution was filtered through a 0.22 μm syringe filter (Millipore, Darmstadt, Germany) and stored at 4° C.
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