- Proteins and Peptides
- Lysates and Cell Lines
Western Blot Procedure
1) Resolve protein samples on a 6% SDS-PAGE gel at 185V for ~1.5 hours.
2) Transfer to PVDF membranes at 25V for ~1.5 hours.
3) Block the membrane with TBST+BSA and goat serum for 1 hour at RT.
4) Dilute primary antibody (NB 100-306) to 1:1,000 in blocking buffer.
5) Incubate membrane overnight at 4 degrees Celcius in diluted anti-ATM-kinase.
6) Wash 3 times ten minutes on a shaker.
7) Incubate membranes with HRP conjugated anti-mouse IgG for 1 hour (RT), diluted in blocking buffer.
8) Wash 3 times ten minutes on a shaker.
9) Add ECL reagent, as per kit directions, and expose for 1-5 seconds.