p38α, MKK6, and Gadd45α proteins were expressed in the bacterial strain BL21(DE3) using the vectors pGEX-4T-1 or pET15b. The 10 amino acid internal deletion mutation of Gadd45α (G45αΔ71–80) was generated by site-directed mutagenesis using the QuikChange kit (Stratagene). For the synthesis of small peptide fragments of Gadd45α, oligonucleotides were synthesized, annealed, and ligated to the pGEX-4T-1 vector. After plasmid transformation, single colonies were grown in culture, and after reaching an A600 of 0.5–1.0 protein expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside. Cultures were further incubated at 14°C overnight. Cells were resuspended in PBS (including 0.5 M NaCl if His-tagged), 0.1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 20,000 × g at 4°C for 20 min. His-tagged proteins were purified with cobalt-charged chelating-Sepharose Fast Flow beads and eluted with 0.3 M imidazole in PBS with 0.5 M NaCl. GST-tagged proteins were purified with Glutathione-Sepharose Fast Flow beads and eluted with 50 mM Tris, pH 8, containing 20 mM glutathione. Proteins were concentrated and washed into PBS using Microcon YM-30 spin columns (Millipore).
Microcon ym 30 spin columns
Manufactured by Merck Group
The Microcon YM-30 spin columns are a type of laboratory equipment used for the concentration and desalting of macromolecular samples, such as proteins, DNA, and RNA. The columns contain a semi-permeable membrane that allows the passage of small molecules and ions while retaining the larger target molecules. Samples are loaded into the column and centrifuged, resulting in the concentration of the target macromolecules in the retentate, and the removal of unwanted smaller components in the filtrate.
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5 protocols using microcon ym 30 spin columns
1
Bacterial Expression and Purification of p38α, MKK6, and Gadd45α
2
Bacterial Expression and Purification of p38α, MKK6, and Gadd45α
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p38α, MKK6, and Gadd45α proteins were expressed in the bacterial strain BL21(DE3) using the vectors pGEX-4T-1 or pET15b. The 10 amino acid internal deletion mutation of Gadd45α (G45αΔ71–80) was generated by site-directed mutagenesis using the QuikChange kit (Stratagene). For the synthesis of small peptide fragments of Gadd45α, oligonucleotides were synthesized, annealed, and ligated to the pGEX-4T-1 vector. After plasmid transformation, single colonies were grown in culture, and after reaching an A600 of 0.5–1.0 protein expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside. Cultures were further incubated at 14°C overnight. Cells were resuspended in PBS (including 0.5 M NaCl if His-tagged), 0.1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 20,000 × g at 4°C for 20 min. His-tagged proteins were purified with cobalt-charged chelating-Sepharose Fast Flow beads and eluted with 0.3 M imidazole in PBS with 0.5 M NaCl. GST-tagged proteins were purified with Glutathione-Sepharose Fast Flow beads and eluted with 50 mM Tris, pH 8, containing 20 mM glutathione. Proteins were concentrated and washed into PBS using Microcon YM-30 spin columns (Millipore).
3
Purification of Key Signaling Proteins
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Recombinant p38α, constitutively active MKK6 (S207E/T211E), ATF2, and truncated NFAT1 (tNFAT1, amino acids 1–350) were purified as described [50 (link)]. In brief, pET15b vectors containing p38α or MKK6 and pGEX-4T1 vectors containing ATF2 or tNFAT1 were expressed in BL21(DE3) cells. After cultures reached an A600 of 0.6–1.0, protein expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG), and the cultures were incubated overnight at 14 °C. For pET15b vectors containing His-tagged fusion proteins p38α or MKK6, cells were resuspended in binding buffer containing 20 mM Tris pH 7.5, 0.5 M NaCl, 20 mM imidazole, and 1 mM phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 20,000 × g for 20 minutes at 4 °C. Proteins were purified with cobalt-charged chelating-Sepharose Fast Flow beads (Amersham Biosciences) and eluted with 0.35 M imidazole in binding buffer. For pGEX-4T1 vectors containing GST-tagged fusion proteins ATF2 or tNFAT1, cells were resuspended in cold PBS and 1 mM phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 20,000 × g for 20 minutes at 4 °C. Proteins were purified with Glutathione Sepharose 4 fast Flow beads (GE Healthcare) and eluted with 10 mM reduced glutathione in cold PBS. Proteins were concentrated and washed into water using Microcon YM-30 spin columns (Millipore).
4
Comparative Genomic Hybridization Analysis
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CGH array was performed using SurePrint G3 Human CGH Bundle (4 × 180K) (Agilent) according to manufacturer instructions. Briefly, 1 μg of genomic DNA corresponding to either a human male control [20] , EndoC-βH2 or EndoC-βH3 cells both at passage 40 was fragmented by heating at 95 °C for 30 min. Fragmented DNAs were labeled with Cy3 (control DNA) and Cy5 (EndoC-βH2/EndoC-βH3 DNA) fluorescent dUTP, respectively, using Genomic DNA Enzymatic Labeling Kit (Agilent Technology). Microcon YM 30 spin columns (Millipore) were used to remove the unincorporated nucleotides and dyes. Hybridizations of labeled DNA to SurePrint G3 Human CGH Bundle (4 × 180K) array (Agilent) were performed in a hybridization oven at 42 C at 20 rpm for 40 h. Hybridized arrays were then washed following the manufacturer's instructions. Microarray slides were scanned on a Nimblegen MS200 Microarray Scanner at a 2 μm resolution. Feature extraction was done with Cytogenomics Software (Agilent). Extracted data were imported and analyzed using Nexus 7.0 (Biodiscovery).
5
Purification of Recombinant p38α and MKK6
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Recombinant p38α and constitutively active MKK6 (S207E/T211E) were purified as previously described (Mittelstadt et al., 2009 (link)). In brief, p38α and MKK6 were expressed in the bacterial strain BL21(DE3) using the vector pET15b. After cultures reached an A600 of 0.5–1.0, protein expression was induced with 0.5 mM isopropyl β-D-thiogalactopyranoside (IPTG) and incubated overnight at 4°C. Cells were resuspended in ice-cold PBS, including 0.5 M NaCl, 0.1% Triton X-100, and 1 mM phenylmethylsulfonyl fluoride, sonicated, and centrifuged at 20,000 g for 20 min at 4°C. Proteins were purified with cobalt-charged chelating–Sepharose Fast Flow beads (GE Healthcare) and eluted with 0.3 M imidazole in PBS with 0.5 M NaCl. Proteins were concentrated and washed into PBS using Microcon YM-30 spin columns (Millipore).
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