Anti-DRAK2抗体(ab8419)

Cross-reaction for peptide antibodies is not uncommon, the band at 42 kDa is specific to DRAK2, but the 70 kDa band is likely to be against a different protein from our own internal testing.

Thank you for your enquiry. It is unnecessary to isolate nuclear proteins before running a western blot. You can detect DRAK2 by loading the Jurkat whole cell lysate directly onto the gel.

Thank you for your enquiry. The larger band is probably a cross-reaction to a non-related protein or a different member of the same family. We would suggest trying longer exposure of the membrane to x-ray film (30 minutes to overnight). It is also possible that the detection enzyme may be inactivated. Sodium azide inactivates horseradish peroxidase (HRP) irreversibly. Also, bacterial contamination also diminishes HRP activity. Polyvinyl wrap from some sources has been found to hide the signal. You could repeat the incubation with chemiluminescence reagents and place the blot between two pieces of acetate transparency film. Western Blot Protocol: 1) Load 20 to 25 microgram of whole cell lysate per lane in an SDS-PAGE mini gel. 2) Run at 20 mA per gel until the dye front is close to the bottom. 3) Transfer the proteins to a nitrocellulose membrane (S&S NCTM) at 250 mA in transfer buffer for 1-4 h, depending on the size of the target protein. 4) Incubate the blot with blocking buffer (5% non-fat dry milk in TBS) overnight at 4°C or 2 hr at room temperature (RT). 5) Incubate the blot with primary antibody (diluted 1:250 to 1:1000 in blocking buffer) for 1 hr in blocking buffer at RT. 6) Wash the blot 3 x 10 min in washing buffer (TBS containing 0.1% Tween 20) with shaking. 7) Incubate blot with anti-rabbit IgG-HRP conjugate (Sigma) (diluted 1:10,000 - 1:2,000 in blocking buffer) for 1 h in blocking buffer at RT. 8) Wash 3 x 10 min in washing buffer with shaking. 9) Drain washing buffer, add ECL solution (Amersham) and develop for 1 min. 10) Expose to X-ray film for 1 to 30 min. TBS: • 125 mM NaCl • 25 mM Tris pH 8.0 • 0.1% Tween 20 SDS/Running Buffer: • 25 mM Tris • 192 mM Glycine • 0.1% SDS Transfer Buffer: • 20 mM Tris • 150 mM Glycine • 20% methanol • 0.038% SDS