Product Name: VERIFY™ Antigen Standard (Transient Over-expression Cell Lysate)
Application: Antigen standard in Western blot, ELISA and other assays
Expression cell line: Human HEK293T cell line
Epitope tag: C-terminal Myc/DDK*
Plasmid: pCMV6-Entry vector
1 vial of 100 µg gene specific transient over-expression cell lysate
1 vial of 100 µg empty vector transfected control cell lysate
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)
100 µg lysate is sufficient for 20~50 reactions. Depending on over-expression level and antibody affinity, some lysates can go as low as 0.1µg per load.
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.
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 reconstituted with 2xSDS Sample Buffer provided. After reconstitution, 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.
The transient over-expression cell lysates are validated with mouse monoclonal anti-myc antibody (TA100010) or anti-DDK antibody (TA100011). See “Validation Data”.
In the rare case where over-expression cell lysates do not pass QC, OriGene will contact customers to provide alternative solutions.
* Peptide sequence of the DDK-tag (Flag®): N-DYKDDDDK-C . Flag® is a registered trademark of Sigma-Aldrich.