||Antigen standard for GDNF family receptor alpha 4 (GFRA4), transcript variant 2 is a lysate prepared from HEK293T cells transiently transfected with a TrueORF gene-carrying pCMV plasmid and then lysed in RIPA Buffer. Protein concentration was determined using a colorimetric assay. The antigen control carries a C-terminal Myc/DDK tag for detection.
||HEK 293T cells
||1 vial of 100 µg gene specific transient over-expression cell lysate in RIPA buffer1 vial of 100 µg empty vector transfected control cell lysate in RIPA buffer1 vial of 250ul 2xSDS Sample Buffer (4% SDS, 125mM Tris-HCl pH6.8, 10% Glycerol, 0.002% Bromphenol blue, 100mM DTT)
||HEK293T cells in 10-cm dishes were transiently transfected with Transfection Reagent and 5ug 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.
||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.
||ELISA, WB, IP. WB: Mix equal volume of lysates with 2X SDS Loading Buffer. Boil the mixture for 10 min before loading (for membrane protein lysates, incubate the mixture at room temperature for 30 min). Load 5 ug lysate per lane.