A procedure entailing affinity, ion exchange, and gel filtration steps was used to purify human UAF1436X, UAF1SLD, UBE2T, the USP1-UAF1 complex, and the ID2 complex9 (link),27 (link). RAD51AP1 and the RAD51AP1DM mutant were expressed in E. coli and purified by GST or MBP tag affinity chromatography and Source S (GE HealthCare) ion exchange chromatography31 (link),32 (link). FANCB-FANCL-FAAP100 (BL100) complex with N-terminally Flag-tagged FANCB was expressed in insect cells using the multi-bacmid provided by Andrew Deans (University of Melbourne, Australia) and purified by Flag tag affinity chromatography and MiniQ (GE Healthcare) ion exchange chromatography11 (link), with an added terminal step of size exclusion in Superdex 200 using buffer A (20 mM Tris-HCl, pH 7.5, 10% glycerol, 0.5 mM EDTA, 0.01% Igepal, 1 mM DTT, and 150 mM KCl). HA-Ubiquitin and UBE1 were purchased from Boston Biochem.
Source s
Manufactured by GE Healthcare
The Source S is a laboratory equipment product manufactured by GE Healthcare. It is designed to serve as a source of radiation for various scientific and medical applications. The device generates and provides a controlled source of radiation, enabling researchers and professionals to conduct experiments, analyses, and other activities that require a reliable and consistent radiation source.
Automatically generated - may contain errors
Lab products found in correlation
Source q, by GE Healthcare (6 mentions)
Superdex 200, by GE Healthcare (3 mentions)
Amicon ultra 15, by Merck Group (2 mentions)
Ha ubiquitin, by R&D Systems (1 mentions)
Miniq, by GE Healthcare (1 mentions)
E coli bl21 de3 cells, by New England Biolabs (1 mentions)
Glutathione agarose resin, by Thermo Fisher Scientific (1 mentions)
C3 emulsiflex, by Roche (1 mentions)
Superdex 75, by GE Healthcare (1 mentions)
Pgex 6p 1 vector, by GE Healthcare (1 mentions)
Protease inhibitor cocktail tablet, by Roche (1 mentions)
Superose 12, by GE Healthcare (1 mentions)
12 protocols using source s
1
Purification of Protein Complexes via Affinity, Ion Exchange, and Gel Filtration
2
Insulin Analog Purification Protocol
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Example 6
The thus-reacted sample was loaded onto a Source S (GE healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and then the insulin analog proteins were eluted in 10 column volumes with a linear gradient from 0% to 100% 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol.
Example 7
Salts were removed from the eluted sample using a desalting column, and the buffer was exchanged with a buffer (10 mM Tris-HCl, pH 7.5). In order to isolate a pure insulin analog from the sample obtained in Example 6, the sample was loaded onto an anion exchange column (Source Q: GE healthcare) equilibrated with 10 mM Tris (pH 7.5) buffer, and the insulin analog protein was eluted in 10 column volumes with a linear gradient from 0% to 100% 10 mM Tris (pH 7.5) buffer containing 0.5 M sodium chloride.
The purity of the thus-purified insulin analog was analyzed by protein electrophoresis (SDS-PAGE,
As a result, each insulin analog was found to have the desired modification in its amino acid sequence.
3
Purification of BRG1 AT-BD and BD
4
Purification of Rad4-Rad23 Complex
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The Rad4–Rad23 and Rad4–Rad23–DNA complexes were prepared as previously described (45 (link)). Briefly, the Hi5 insect cells co-expressing the Rad4–Rad23 complex were harvested 2 days after infection. After lysis, the proteins were purified using His-Select Nickel agarose resin (Sigma) and anion exchange chromatography (Source Q, GE Healthcare), followed by thrombin digestion and cation exchange (Source S, GE Healthcare) and gel-filtration (Superdex200, GE Healthcare) chromatography. The final sample was concentrated by ultrafiltration to ∼13 mg ml−1 in 5 mM bis–tris propane–HCl (BTP-HCl), 800 mM NaCl, 5 mM dithiothreitol (DTT), pH 6.8.
5
Purification and Characterization of Insulin Analogues
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Example 6
The sample thus reacted was loaded onto a Source S (GE healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and then the insulin analogue proteins were eluted in 10 column volumes with a linear gradient from 0% to 100% 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol.
Example 7
Salts were removed from the eluted sample using a desalting column, and the buffer was exchanged with a buffer (10 mM Tris-HCl, pH 7.5). In order to isolate pure insulin analogues from the sample obtained in Example 6, the sample was loaded onto an anion exchange column (Source Q: GE healthcare) equilibrated with 10 mM Tris (pH 7.5) buffer, and the insulin analogue protein was eluted in 10 column volumes with a linear gradient from 0% to 100% 10 mM Tris (pH 7.5) buffer containing 0.5 M sodium chloride.
Purity of the insulin analogue thus purified was analyzed by protein electrophoresis (SDS-PAGE) and high pressure chromatography (HPLC), and modifications of amino acids were identified by peptide mapping and molecular weight analysis of each peak.
As a result, each insulin analogue was found to have the desired change in its amino acid sequence.
6
Insulin Analog Purification by Ion-Exchange
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Example 4
The refolded sample was loaded onto a Source S (GE healthcare) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol, and then the insulin analog proteins were eluted in 10 column volumes with a linear gradient from 0% to 100% 20 mM sodium citrate (pH 2.0) buffer containing 0.5 M potassium chloride and 45% ethanol.
7
Insulin Analog Purification Protocol
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Example 4
The refolded sample was combined bound to Source S (GE healthcare, Inc.) column equilibrated with 20 mM of sodium citrate contained in the site is equilibrated with 45% ethanol (pH 2.0) buffer containing 4% ethanol. The insulin analog protein was then eluted with 10 column volume of linear gradient using 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol and 0.5 M of potassium chloride so that the concentration is 0% to 100%.
8
Purification of Rad4-Rad23 Complex
9
Purification of Recombinant Pellino Proteins
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GST- and His6-Pellinos
were expressed in Escherichia coli BL21(DE3) pLys
cells and purified essentially as described previously,20 (link) with the following modifications. Glutathione
agarose beads were incubated with GST-Pellinos, washed three times
in 50 mM sodium phosphate and 300 mM NaCl (pH 7.4), and resuspended
in 20 mM sodium phosphate and 150 mM NaCl (pH 7.4) (PBS). To remove
the His6 tag, His6-Pellinos with TEV protease26 (∼100 μg for every 5 mg of His6-Pellino) were dialyzed against 10 mM HEPES, 300 mM NaCl,
and 5 mM β-mercaptoethanol (βME) (pH 7.0). Dialysis was
performed overnight at 4 °C for Pellino1 and -2 and at 22 °C
for Pellino3a and -3b. Digested, untagged Pellinos were purified by
cation exchange chromatography (Source S, GE Healthcare) at pH 6 (Pellino1,
-2, and -3b) or pH 5.5 (Pellino3a), followed by size exclusion chromatography
(Superose 12, GE Healthcare) in 10 mM HEPES, 150 mM NaCl, and 5 mM
βME (pH 7.0).
were expressed in Escherichia coli BL21(DE3) pLys
cells and purified essentially as described previously,20 (link) with the following modifications. Glutathione
agarose beads were incubated with GST-Pellinos, washed three times
in 50 mM sodium phosphate and 300 mM NaCl (pH 7.4), and resuspended
in 20 mM sodium phosphate and 150 mM NaCl (pH 7.4) (PBS). To remove
the His6 tag, His6-Pellinos with TEV protease26 (∼100 μg for every 5 mg of His6-Pellino) were dialyzed against 10 mM HEPES, 300 mM NaCl,
and 5 mM β-mercaptoethanol (βME) (pH 7.0). Dialysis was
performed overnight at 4 °C for Pellino1 and -2 and at 22 °C
for Pellino3a and -3b. Digested, untagged Pellinos were purified by
cation exchange chromatography (Source S, GE Healthcare) at pH 6 (Pellino1,
-2, and -3b) or pH 5.5 (Pellino3a), followed by size exclusion chromatography
(Superose 12, GE Healthcare) in 10 mM HEPES, 150 mM NaCl, and 5 mM
βME (pH 7.0).
10
Insulin Analog Purification Protocol
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Example 6
The Reaction-Completed Sample was Again Combined with Source S (GE healthcare, Inc.) column equilibrated with 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol. The insulin analog protein was then eluted with 10 column volume of linear gradient using 20 mM sodium citrate (pH 2.0) buffer containing 45% ethanol and 0.5 M of potassium chloride so that the concentration is 0% to 100%.
Example 7
The salt was removed from a sample eluted with a desalting column, and replaced with a buffer solution (10 mM Tris-HCl, pH 7.5). In order to separate a pure insulin analog from the sample obtained in Example 6, the sample was combined with an anion exchange column (Source Q: GE healthcare, Inc.) equilibrated with 10 mM tris (pH 7.5) buffer solution. The insulin analog protein was then eluted with 10 column volume of linear gradient using 10 mM tris (pH 7.5) buffer solution containing 0.5M sodium chloride so that the concentration is 0% to 100%.
The purity of the purified insulin analog were analyzed using protein electrophoresis (SDS-PAGE) and high-pressure chromatography (HPLC), and the amino acid changes were confirmed through a peptide mapping and a molecular weight analysis of each peak.
As a result, it was confirmed that the amino acid sequence was changed according to a desired purpose of respective insulin analog.
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