Enzymes for molecular biology were from New England Biolabs. TOP10 ultracompetent cells, the pYES2CT vector, anti-V5-HRP, molecular weight markers and native PAGE gels and reagents were from Life Technologies. Glass econocolumns and the DC protein assay kit were from BioRad. Denatured sheared salmon sperm DNA and reagents for assays of esterase and acyltransferase activity were from Sigma, except p-nitrophenyl hexanoate from Ark chemicals. HisTrap columns, PD-25 gel filtration columns, size-exclusion columns, protein chromatography standards and nitrocellulose membrane were from GE Healthcare. Precast acrylamide gels were from NuSep. LumiGLO chemiluminescence reagents were from Cell Signaling Technology.
Size exclusion columns
Manufactured by GE Healthcare
Size exclusion columns are laboratory instruments used for the separation and purification of macromolecules, such as proteins, peptides, and nucleic acids, based on their size and molecular weight. These columns contain a porous stationary phase that allows smaller molecules to penetrate into the pores, while larger molecules pass through the column more rapidly. This size-based separation technique enables the isolation and concentration of target analytes from complex mixtures.
Automatically generated - may contain errors
Lab products found in correlation
Histrap column, by GE Healthcare (4 mentions)
Kta pure system, by GE Healthcare (3 mentions)
Ultrasonic processor, by Qsonica (2 mentions)
Nanodrop 1000 uv vis spectrometer, by Thermo Fisher Scientific (2 mentions)
Molecular weight marker, by Thermo Fisher Scientific (1 mentions)
Nitrocellulose membrane, by GE Healthcare (1 mentions)
Pyes2 ct vector, by Thermo Fisher Scientific (1 mentions)
Anti v5 hrp, by Thermo Fisher Scientific (1 mentions)
Glass econocolumns, by Bio-Rad (1 mentions)
Enzymes for molecular biology, by New England Biolabs (1 mentions)
Dc protein assay kit, by Bio-Rad (1 mentions)
4 protocols using size exclusion columns
1
Molecular Biology Enzyme Purification
2
Recombinant Protein Purification Protocol
3
Recombinant Expression of GEPIIs
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Recombinant expression of GEPIIs was performed using pETM-11 bacterial expression vectors. Proteins were expressed in E. coli BL-21 (DE3) cells. At an OD600 of 0.8, protein expression was induced by adding 1 mM β-d -1-thiogalactopyranoside (IPTG) and cells were incubated at room temperature. After 4 h cells were pelleted and cells were re-suspended in 20 ml of lysis buffer. Then cells were lysed by sonication and cleared by centrifugation. Proteins were purified using a 5 ml HisTrap column (GE Healthcare, Vienna, Austria) for immobilized metal affinity chromatography on an ÄKTA pure system (GE Healthcare, Vienna, Austria) at room temperature. HisTrap columns were equilibrated using lysis buffer, E. coli lysates were applied on the columns and washed with washing buffer. Proteins were eluted with purification buffer and the His6-Protein A tag was cleaved overnight at 4 °C using 2% (w/w) of 1 mg ml-1 recombinant His-tagged TEV protease. Processed proteins were re-purified from the fusion tags and TEV protease at room temperature using size exclusion columns (16/600 200 pg, GE Healthcare) on an ÄKTA pure system (GE Healthcare). Subsequently proteins were eluted using elution buffer.
4
Protein Purification by Affinity and Size Exclusion Chromatography
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Protein
expression was induced by adding 1 mM β-d -1-thiogalactopyranoside
(IPTG) at an OD600 of 0.8. Cell pellets were resuspended in 20 mL
lysis buffer, followed by sonication (QSONICA Ultrasonic Processor;
12 min, 50% amplitude, 1 s on/off) for cell lysis. Centrifugation
at 12 000 rpm for 45 min at 4 °C (Sorvall LYNX 6000) and
filtration (0.45 μm cellulose acetate syringe filters) was used
to clear the lysates. 5 mL HisTrap columns (GE Healthcare, Vienna,
Austria) for immobilized metal affinity chromatography on an ÄKTA
pure system (GE Healthcare, Vienna, Austria) were used to purify the
proteins. HisTrap columns were equilibrated using lysis buffer. Cleared E. coli lysates were applied to the columns at a flow rate
of 2 mL min–1 and contaminants were removed using
washing buffer. Finally, proteins were eluted with purification buffer.
Proteins were further purified at room temperature using size exclusion
columns (10/300 200 pg, GE Healthcare) on an ÄKTA pure system
(GE Healthcare) with SEC buffer. Finally, protein concentration was
calculated using absorbance at 280 nm, determined by NanoDrop 1000
UV/vis spectrometer (Thermo Fisher Scientific, Vienna, Austria).
expression was induced by adding 1 mM β-
(IPTG) at an OD600 of 0.8. Cell pellets were resuspended in 20 mL
lysis buffer, followed by sonication (QSONICA Ultrasonic Processor;
12 min, 50% amplitude, 1 s on/off) for cell lysis. Centrifugation
at 12 000 rpm for 45 min at 4 °C (Sorvall LYNX 6000) and
filtration (0.45 μm cellulose acetate syringe filters) was used
to clear the lysates. 5 mL HisTrap columns (GE Healthcare, Vienna,
Austria) for immobilized metal affinity chromatography on an ÄKTA
pure system (GE Healthcare, Vienna, Austria) were used to purify the
proteins. HisTrap columns were equilibrated using lysis buffer. Cleared E. coli lysates were applied to the columns at a flow rate
of 2 mL min–1 and contaminants were removed using
washing buffer. Finally, proteins were eluted with purification buffer.
Proteins were further purified at room temperature using size exclusion
columns (10/300 200 pg, GE Healthcare) on an ÄKTA pure system
(GE Healthcare) with SEC buffer. Finally, protein concentration was
calculated using absorbance at 280 nm, determined by NanoDrop 1000
UV/vis spectrometer (Thermo Fisher Scientific, Vienna, Austria).
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