ITCH (NM_031483) Human Over-expression Lysate

The lysate is shipped with dry ice. Upon receiving, store the sample at -80°C. After dilution, the protein sample should be aliquoted and stored at -80°C for long term storage. Avoid repeated freeze-thaw cycles. Lysate samples can be diluted with 2 x SDS Sample Buffer provided. Lysate samples are stable for 12 months from the date of receipt when stored at -80°C.

HEK293T cells in 10-cm dishes were transiently transfected with Leading Biology Transfection Reagent (TF002V) and 5ug Leading Biology GoldORF 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, 1 x Proteinase 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 -20°C before shipping.