HeLa Nuclear Cell Lysate

Human

SDS-PAGE, Western Blot

Western Blot (1:100-1:2000)

Ready-to-use nuclear extracts are especially prepared as positive controls for separation by SDS-PAGE and subsequent western blot analysis. Nuclear extracts are supplied in denaturing buffer without dissociating agents. Heat nuclear extract to 95C for 5 minutes and rapidly cool. If dissociating conditions are desired add reducing agent prior to heating. The recommended loading volume per lane is 10-30 ul depending on the size format of your gel.

Cell

Nuclear

The cells were grown in Dulbecco's medium supplemented with 10% fetal bovine serum. Cells were washed with PBS and then incubated on ice in modified RIPA buffer, containing 150 mM sodium chloride, 50 mM Tris HCl, pH 7.4, 1 mM EDTA, 1.0% NP-40, 0.5% sodium deoxycholic acid , 0.1% SDS and 0.01% (w/v) sodium azide to lyse the cells. Protein integrity was ensured using a cocktail of protease inhibitors with broad specificity for the inhibition of aspartic, cysteine, and serine proteases as well as aminopeptidases (0.1 mM AEBSF HCl, 0.08 uM Aprotinin, 5 uM Bestatin, 1.5 uM E-64, 2 uM Leupeptin Hemisulfate, 1 uM Pepstatin A). Phosphatase inhibitors 1 mM NaF and 1 mM Na3VO4 were also added. Cell debris was removed by centrifugation. Protein concentration was determined by a modified Lowry assay using a commercially available kit. Protein concentration was adjusted to 2 mg/ml and then an equal volume of 2X SDS-PAGE sample buffer was added.

Multi-step

1X SDS-PAGE Sample Buffer (62.5 mM Tris HCl, 2% SDS, 10% Glycerol and 0.005% bromophenol blue, pH 6.8), 10% (v/v) Glycerol

No Preservative

Please contact technical services for concentration.

The product is shipped with dry ice or equivalent. Upon receipt, store it immediately at the temperature recommended below.

Store at -70C. Avoid freeze-thaw cycles.