Ssb or Ssb-GFP was purified from E. coli cells encoding Ssb N-terminally fused with a cleavable His6-SUMO tag. Cells were lysed in buffer A (40 mM Hepes KOH pH 7.4, 150 mM KCl, 5 mM MgCl2, 5% glycerol, 1 mM PMSF, 10 mM β-mercaptoethanol, 1 mM ETDA, DNaseI) and cleared lysates were mixed with nickel–iminodiacetic acid (Ni-IDA, Protino, Macherey-Nagel). After extensive washing with buffer A, His6-SUMO-Ssb was eluted with buffer A containing 250 mM Imidazole. His6-SUMO was removed by treatment with His6-ULP protease during overnight dialysis against buffer A. His6-ULP, His6-SUMO and uncleaved His6-SUMO-Ssb were removed by passing the eluate through a second Ni-IDA column. Ssb was loaded onto a HiLoad 16/60 Superdex 75 gel filtration column (GE Healthcare Life Sciences) and gel filtration was performed in buffer B (40 mM Hepes KOH pH 7.4, 50 mM KCl, 5 mM MgCl2, 5% glycerol, 10 mM β-mercaptoethanol) containing 5 mM ATP. Aliquots were stored at - 80 °C.
Protino
Manufactured by Macherey-Nagel
Protino is a lab equipment product from Macherey-Nagel that is used for protein purification. It serves as a tool for the isolation and separation of proteins from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Ni ida, by Macherey-Nagel (1 mentions)
Superdex 75 hiload 16 60 gel filtration column, by GE Healthcare (1 mentions)
Lyophilization, by Labconco (1 mentions)
10 dg desalting column, by Bio-Rad (1 mentions)
Steriflip filter, by Merck Group (1 mentions)
Sigmafast protease inhibitor, by Merck Group (1 mentions)
Completetm, by Merck Group (1 mentions)
Amicon filter unit, by Merck Group (1 mentions)
Resourcetm q, by GE Healthcare (1 mentions)
4 protocols using protino
1
Purification and Characterization of Ssb Protein
2
Purification of Histidine-Tagged Protein
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The pellet was transferred
to PBS buffer and centrifuged for 10 min at 4300 rcf. The resulting
pellet was lysed in 30 mL of lysis buffer, referred to hereafter as
buffer B (20 mM bicine, pH 8.5, 500 mM NaCl, 10 mM imidazole), supplemented
with one tablet of EDTA-free SigmaFast Protease Inhibitor (Sigma-Aldrich),
5 mM PMSF, and 2 mg of lysozyme. Without incubation, the resuspension
was lysed with an Avestin C3 homogenizer followed by a 20 min centrifugation
at 24,000 rcf at 4 °C. The supernatant was filtered through a
40 μm Steriflip filter (Millipore) and loaded onto a 5 mL NiNTA
column (Protino, Machery Nagel) connected to an Akta purifier preequilibrated
with buffer B. The column was washed with 50 mL (10 column volumes)
of 20 mM bicine (pH 8.5), 500 mM NaCl, 10 mM imidazole, 10 mM β-ME.
The protein was eluted with 20 mM bicine (pH 8.5), 500 mM NaCl, 250
mM imidazole, 10 mM β-Me. Imidazole was removed by exchanging
against 20 mM bicine (pH 8.5), 500 mM NaCl, with a 10DG desalting
column (BioRad). For purposes of lyophilization, the protein was directly
exchanged against 20 mM bicine (pH 8.5) and 100 mM trehalose, followed
by flash freezing with liquid N2 and lyophilization (Labconco)
overnight. Typical protein yields are 800 μM (3 mL total) from
a 1 L culture, with a purity of ∼98% by SDS–PAGE and
LC–MS (ESI-TOF) (6224 TOF and 1200 series HPLC, Agilent Technologies).
to PBS buffer and centrifuged for 10 min at 4300 rcf. The resulting
pellet was lysed in 30 mL of lysis buffer, referred to hereafter as
buffer B (20 mM bicine, pH 8.5, 500 mM NaCl, 10 mM imidazole), supplemented
with one tablet of EDTA-free SigmaFast Protease Inhibitor (Sigma-Aldrich),
5 mM PMSF, and 2 mg of lysozyme. Without incubation, the resuspension
was lysed with an Avestin C3 homogenizer followed by a 20 min centrifugation
at 24,000 rcf at 4 °C. The supernatant was filtered through a
40 μm Steriflip filter (Millipore) and loaded onto a 5 mL NiNTA
column (Protino, Machery Nagel) connected to an Akta purifier preequilibrated
with buffer B. The column was washed with 50 mL (10 column volumes)
of 20 mM bicine (pH 8.5), 500 mM NaCl, 10 mM imidazole, 10 mM β-ME.
The protein was eluted with 20 mM bicine (pH 8.5), 500 mM NaCl, 250
mM imidazole, 10 mM β-Me. Imidazole was removed by exchanging
against 20 mM bicine (pH 8.5), 500 mM NaCl, with a 10DG desalting
column (BioRad). For purposes of lyophilization, the protein was directly
exchanged against 20 mM bicine (pH 8.5) and 100 mM trehalose, followed
by flash freezing with liquid N2 and lyophilization (Labconco)
overnight. Typical protein yields are 800 μM (3 mL total) from
a 1 L culture, with a purity of ∼98% by SDS–PAGE and
LC–MS (ESI-TOF) (6224 TOF and 1200 series HPLC, Agilent Technologies).
3
Purification of ParB Protein from E. coli
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ParB protein production was performed in E. coli BL21 pLysS via the pET-16b vector-based system. Cells were grown in LB at 37 °C; gene expression was induced adding 1 mM IPTG following growth for 12 h at 18 °C. Subsequently, cells were suspended in washing buffer (50 mM Tris-HCl pH 7.4; 100 mM NaCl; 5 mM; MgCl2; 1 mM dithiothreitol) containing EDTA-free proteinase inhibitor (cOmpleteTM, Sigma) and DNAseI, and lysed using a high-pressure cell homogenizer. Cell debris and membranes were removed by centrifugation at 4 °C, 1700 g for 20 min, and 150,000 × g for 45 min, respectively. Thereupon, batch purifications of His-tagged protein were performed under native conditions using Ni-NTA agarose (Protino®, Macherey-Nagel) according to manufacturer’s instruction. In brief, the equilibrated gel was incubated with clarified lysate for 60 min at 4 °C under gentle agitation and washed twice in washing buffer containing 80 mM imidazole. Proteins were eluted in three steps using washing buffer with an imidazole concentration of 300 mM, concentrated via Amicon filter units (Merck) and further purified by applying size exclusion chromatography using an ÄKTApurifier system with a SuperdexTM 200 gel filtration column (GE Healthcare Life Sciences).
4
Expression and Purification of HtpG
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HtpG and HtpG E34A were expressed as C-terminal His 10 -fusions (Graf et al., 2009) in E. coli MC4100 DhtpG:kan, and purified by Ni-IDA-chromatography (Protino, Macherey-Nagel) and anion-exchange chromatography (Resource TM Q, GE Healthcare). The purified proteins were checked to be nucleotide-free by anion-exchange chromatography (Resource TM Q) and by UV detection by 254 nm.
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