- Proteins and Peptides
- Lysates and Cell Lines
Western blot Protocol:
1) Transfer onto PVDF membrane
2) Block in 3% (w/v) skimmed milk in TBS-T (0.05% Tween 20) for 1 hr at room temperature with agitation (can be blocked overnight at 4C without agitation).
3) Incubate NB100-1194 (2ug/ml) for 1 hr at room temperature with agitation.
4) Wash with TBS-T three times
5) Incubate with secondary antibody (1:20,000 or higher). We use Jackson or Rockland anti-goat secondary (affinity purified and with minimal cross-reactivity with Human and rodent serum proteins) for 1 hr at room temperature with agitation.
6) Wash with TBST three times.
7) Apply the detection reagent of choice in accordance with the manufacturers instructions (We use ECL-plus (Amersham) rather than ECL, which is more sensitive for detection.)
*All antibodies are incubated in blocking buffer.
*QC testing was done under reducing conditions (with beta mercaptoethanol at 100C).