Home Over-expression Lysates RELA Lysate|
RELA Over-expression Lysate Product
RELA Activity Verified in a DNA-binding Assay
Also for RELA (NM_021975)
|Expression cell line:
||Human HEK293T cell line
|Expression cDNA Clone:
||TrueORF Clone RC220780|
|Epitope tag:||C-terminal Myc/DDK*|
|Detection Antibodies:||Anti-DDK Antibody (TA50011)|
||Western Blot experiments.
Left-Control; Right -Over-expression Lysate
|Accession Number:||NM_021975 NP_068810|
|Other Names:||RELA,NFKB3; p65
|Predicted MW:||60 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 0.5 ml 2xSDS Sample Buffer (4%SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromphenol blue, 5% b-mercaptoethanol)
|Storage:||The lysate is shipped with dry ice. Upon receiving, store the sample at -20ºC. Lysate samples are stable for 12 months from date of receipt when stored at -20ºC. Avoid repeated freeze-thaw cycles. |
Lysate samples can be diluted with 2xSDS Sample Buffer provided. After dilution, the protein sample should be aliquoted and stored at -20ºC for long term storage. Prior to SDS-PAGE fractionation, boil the lysate for 5 minutes.
|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.). Cell lysates were aliquoted and stored at -20ºC before shipping.
||Transcription FactorsDruggable Genome
||MAPK signaling pathwayChemokine signaling pathwayApoptosisToll-like receptor signaling pathwayNOD-like receptor signaling pathwayRIG-I-like receptor signaling pathwayMore Pathways >>
*: DDK-tag is the same as FLAG tag. Flag® is a registered trademark of Sigma-Aldrich
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.