Recombinant Human CHAT HEK293T cell lysate

• Cat.No.: CHAT-175HCL
• Product Overview: Human CHAT derived in Human HEK293T cell line. The cell lysate is provided in RIPA buffer.

Specification

• Species: Human
• Source: HEK293
• Tag: DDK&Myc
• Description: This gene encodes an enzyme which catalyzes the biosynthesis of the neurotransmitter acetylcholine. This gene product is a characteristic feature of cholinergic neurons, and changes in these neurons may explain some of the symptoms of Alzheimer disease. Mutations in this gene are associated with congenital myasthenic syndrome associated with episodic apnea. Multiple transcript variants encoding different isoforms have been found for this gene, and some of these variants have been shown to encode more than one isoform.
• Molecular Mass: 82.4 kDa
• Components: 1 vial of 100 µg gene specific transient over-expression cell lysate in RIPA buffer;1 vial of 100 µg empty vector transfected control cell lysate in RIPA buffer;1 vial of 250ul 2xSDS Sample Buffer (4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromphenol blue, 100mM DTT).
• Preparation method: HEK293T cells in 10-cm dishes were transiently transfected with MegaTran Transfection Reagent and 5ug TrueORF cDNA plasmid. Transfected cells were cultured for 48hrs before collection. The cells were lysed in modified RIPA buffer (25mM Tris-HCl pH7.6, 150mM NaCl, 1% NP-40, 1mM EDTA, 1xProteinase inhibitor cocktail mix, 1mM PMSF and 1mM Na3VO4), and then centrifuged to clarify the lysate. Protein concentration was measured by BCA kit. Cell lysates were aliquoted and stored at -20 centigrade before shipping.

Gene Information

• Gene Name: CHAT choline O-acetyltransferase [ Homo sapiens ]
• Official Symbol: CHAT
• Synonyms: CHAT; choline O-acetyltransferase; choline acetyltransferase; choline acetylase; acetyl CoA:choline O-acetyltransferase; CMS1A; CMS1A2; CHOACTASE
• Gene ID: 1103
• mRNA Refseq: NM_020549
• Protein Refseq: NP_065574
• MIM: 118490
• UniProt ID: P28329
• Chromosome Location: 10q11.2
• Pathway: Acetylcholine Neurotransmitter Release Cycle, organism-specific biosystem; Acetylcholine Synthesis, organism-specific biosystem; Biogenic Amine Synthesis, organism-specific biosystem; Cholinergic synapse, organism-specific biosystem; Glycerophospholipid metabolism, organism-specific biosystem; Glycerophospholipid metabolism, conserved biosystem; Neuronal System, organism-specific biosystem
• Function: choline O-acetyltransferase activity; choline binding; transferase activity, transferring acyl groups

Citation

Epigenetic quantification of immunosenescent CD8 + TEMRA cells in human blood

Journal: Aging Cell    PubMed ID: 35397197    Data: 2022/4/9

Authors: Ahto Salumets, Liina Tserel, P?rt Peterson

Article Snippet:Two fragments of immunodominant regions of CMV antigen pp150 were cloned into pNanoLuc vector, and LIPS was performed as reported earlier (Haljasm?gi et al., ).Two fragments of immunodominant regions of CMV antigen pp150 were cloned into pNanoLuc vector, and LIPS was performed as reported earlier (Haljasm?gi et al., ).. The HEK293 cell lysates containing NanoLuc‐fusion proteins (0.5–1 × 10 6 luminescence units; LU) were incubated with plasma samples and Protein G Sepharose beads (Creative BioMart) to capture antibodies (in 1:40 dilution).. After washing, the substrate was added (Nano‐Glo? Luciferase Substrate, Promega), and luminescence was measured in VICTOR X Reader (PerkinElmer Life Sciences).After washing, the substrate was added (Nano‐Glo? Luciferase Substrate, Promega), and luminescence was measured in VICTOR X Reader (PerkinElmer Life Sciences).

Longitudinal proteomic profiling reveals increased early inflammation and sustained apoptosis proteins in severe COVID-19

Journal: Scientific Reports    PubMed ID: 33239683    Data: 2020/11/25

Authors: Liis Haljasm?gi, Ahto Salumets, P?rt Peterson

Article Snippet:SARS-CoV-2 S (S1 aa 1–680 and S2 aa 820–1211), S1 RBD (aa 329–538), and N (aa 2–419) fragments were cloned into pNanoLuc vector, and LIPS was performed as reported .SARS-CoV-2 S (S1 aa 1–680 and S2 aa 820–1211), S1 RBD (aa 329–538), and N (aa 2–419) fragments were cloned into pNanoLuc vector, and LIPS was performed as reported .. The HEK293 cell lysates containing NanoLuc‐fusion proteins (0.5—1 × 10 6 luminescence units; LU) were incubated with plasma samples and Protein G Sepharose beads (Creative BioMart) to capture antibodies (in 1:40 dilution).. After washing, substrate was added (Nano-Glo? Luciferase Substrate, Promega), and luminescence was measured in VICTOR X Reader (PerkinElmer Life Sciences).After washing, substrate was added (Nano-Glo? Luciferase Substrate, Promega), and luminescence was measured in VICTOR X Reader (PerkinElmer Life Sciences).

Supraphysiological levels of GDF 11 induce striated muscle atrophy

Journal: EMBO Molecular Medicine    PubMed ID: 28270449    Data: 2017/3/7

Authors: David W Hammers, Melissa Merscham‐Banda, H Lee Sweeney

Article Snippet:At day 6, myotube cultures were treated with various doses of the recombinant ligands (listed above) for 1 h ( n = 4), and SMAD phosphorylation was assayed as mentioned above.At day 6, myotube cultures were treated with various doses of the recombinant ligands (listed above) for 1 h ( n = 4), and SMAD phosphorylation was assayed as mentioned above.. For ActRIIB antibody (anti‐ActRIIB) assays, HEK293T cells were treated with 0 or 100 nM anti‐ActRIIB (Creative BioMart, murinized version of BYM338 antibody; Lach‐Trifilieff et al , ) overnight prior to ligand addition, lysing, and AlphaLISA evaluation, as described above.

Relative p‐SMAD2/3 (left) and p‐SMAD1/5/8 (right) response, evaluated by AlphaLISA signal, of HEK293T cells after 1‐h exposure to recombinant myostatin (Mstn), activin A, GDF11, TGFβ, and BMP2. Relative p‐SMAD2/3 response of C2C12 myoblasts (left) and myotubes (right) after 1‐h exposure to Mstn, activin A, GDF11, and TGFβ. Phosphorylation of SMAD2 and SMAD3 in differentiated C2C12 myotubes following stimulation with recombinant Mstn or GDF11 for 30 and 60 min, as detected by immunoblotting. Equal loading is verified by Ponceau Red staining. EC 50 values (in nM) for p‐SMAD2/3 and p‐SMAD1/5/8 responses of HEK293T, C2C12 myoblasts, and C2C12 myotubes to the above listed ligands, as well as p‐SMAD1/5/8 response to BMP4, BMP6, and BMP7. Data information: Values are displayed as mean ± SEM; n = 4 for all data points.

A, B Significant shifts in the response of HEK293T cells to Mstn, GDF11, and activin A are observed with the addition of ActRIIB antibody (anti‐ActRIIB), when 0 or 100 nM anti‐ActRIIB was applied to HEK293T cells overnight prior to ligand addition and AlphaLISA evaluation. Note that the 0 nM antibody treatment panel is the un‐normalized form of Fig A left. EC 50 values (in nM) are listed for the antibody data in (B). C p‐SMAD1/5/8 response of C2C12 myoblasts (left) and myotubes (right) in response to TGFβ family member ligands. All cells were stimulated with ligand 1 h prior to lysis and evaluation by AlphaLISA. Data Information: Values are displayed as mean ± SEM; n = 4 for all data points.