NF-kB p65 (RELA) (NM_021975) Human Over-expression Lysate

CAT#: LC402895

RELA HEK293T cell transient overexpression lysate (as WB positive control)

Product Images

RELA activity in the over-expression lysate (LY402895) and a vector-transfected control lysate was measured in a colorimetric DNA-binding assay. Double-stranded oligonucleotide containing the RELA consensus DNA-binding sequence was incubated with dilutions of the over-expression lysate and RELA bound to the oligo was captured onto the surface of a microtiter plate. After washing, bound RELA was detected with an anti-RELA primary antibody followed by an HRP-labeled secondary antibody. After initial color development, the reaction was quenched and the color intensity was measured at 450nm. The data show high levels of RELA DNA binding activity in the over-expression lysate, but almost no DNA-binding activity in the control lysate. Overexpression cell lysates are prepared from HEK293T cells transfected with RC220780 using transfection reagent MegaTran 2.0 (Cat# TT210002).

Specifications

Product Data

• Species: Human
• Applications: WB
• Expression Host: HEK293T
• Expression cDNA Clone or AA Sequence: TrueORF Clone RC220780
• Tag: C-Myc/DDK
• Detection Antibodies: Clone OTI4C5, Anti-DDK (FLAG) monoclonal antibody (TA50011-100)
• Accession Number: NM_021975, NP_068810
• Other Names: CMCU; NFKB3; p65
• Predicted MW: 60 kDa
• Components: 1 vial of 20 ug lyophilized gene specific transient over-expression cell lysate
• Storage: The lysate can be shipped at ambient temperature. Upon receiving, store the sample at -20°C. Lysate samples can be reconstituted with SDS Sample Buffer. Avoid repeated freeze-thaw cycles after reconstitution. Lysate samples are stable for 12 months from date of receipt when stored at -20°C.
• Preparation: HEK293T cells in 10-cm dishes were transiently transfected with MegaTran Transfection Reagent (TT200002) 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 (Sigma), 1mM PMSF and 1mM Na3VO4), and then centrifuged to clarify the lysate. Protein concentration was measured by BCA kit (Thermo Scientific Inc.). To facilitate transportation and protein, the products are supplied as lyophilized proteins.

Reference Data

• RefSeq: NP_068810
• Locus ID: 5970
• Cytogenetics: 11q13.1
• Protein Families: Druggable Genome, Transcription Factors
• Protein Pathways: Acute myeloid leukemia, Adipocytokine signaling pathway, Apoptosis, B cell receptor signaling pathway, Chemokine signaling pathway, Chronic myeloid leukemia, Cytosolic DNA-sensing pathway, Epithelial cell signaling in Helicobacter pylori infection, MAPK signaling pathway, Neurotrophin signaling pathway, NOD-like receptor signaling pathway, Pancreatic cancer, Pathways in cancer, Prostate cancer, RIG-I-like receptor signaling pathway, Small cell lung cancer, T cell receptor signaling pathway, Toll-like receptor signaling pathway