Load 5-10ul of the molecular weight standard markers to one well.Heat the samples for 5 min at 95☌, centrifuge and load each sample into individual wells of the stacking gel.Prepare cell lysates by mixing with loading buffer as follows (10-30ug/well for transfected cells, 100-200ug/well for non- transfected cells or tissue extracts).Put the gel into the tank and fill with the electrophoresis running buffer. Choose the appropriate percentage precast gel and remove the top comb and bottom green plastic film or manufacture the gel according to the expected molecular weight of the antigen ( Appendix I).Molecular weight standard markers (Panreac).10X Tris-Glycine buffer (Thermo Scientific 28363) (Transfer buffer: 10x Tris-Glycine Buffer + 20% Methanol in distilled water).10X Electrophoresis running buffer (SIGMA T7777) (Electrophoresis running buffer: 10x Tris-Glycine-SDS Buffer in distilled water).LI-COR Odyssey Imaging System (Model: CLx).Intercept PBS Blocking Buffer (LI-COR Biosciences, P/N: 927-70001).Phosphate buffered saline (PBS, homemade).IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor 680 (Thermo Fisher polyclonal anti mouse/rat/rabbit) Ref: A21057, A21096 and A21076).Tetramethylethyllenediamine (1,2-TEMED - MERCK).30% Acrilamide-Bisacrilamide solution (Bio-Rad 161-0158).

Mini-PROTEAN TGX Precast Gel (Bio-Rad #4561033).

Home > Protocols > Odyssey Western Blotting protocol (OdWB) Odyssey Western Blotting protocol (OdWB) Protocol created by Lorena Maestre - Monoclonal Antibodies Unit, Centro Nacional de Investigaciones Oncológicas Figure 1.

Licor western blot