Wash the blotting membrane for 10 mins by shaking in PBS + 0.1% Tween-20.It can be done by cutting the membrane or re-staining it after the scanning. Use another primary antibody as loading control, choose one that has a different molecular weight from your protein of interest (e.g. Ensure the volume of the antibody solution is enough to fully cover the membrane. Incubate the membrane with the primary antibody for 1 hour at room temperature.Incubate the membrane with Intercept Blocking Buffer and shake overnight at 4☌ or for two hours at 37☌ to block non-specific binding to the membrane.Fill the tank with the transfer buffer and connect it to the PowerPac power supply for 1 hour at 95V (alternative: overnight at 35V, Remove the cassette and open.Place it into the electrophoresis blotting module. Remove the air bubbles by gently rolling a Pasteur pipette over the “sandwich”. Prepare the blotting “sandwich” using the gel holding cassette.Cut the PDVF membrane to size and activate it in the blotting membrane in methanol (1min), wash in distilled water (5min) and finally equilibrate in transfer buffer (5 min).Open the gel cassette and extract the gel, place it gently into the transfer buffer for 5 min.Connect the tank to the PowerPac power supply and run the gel at 100 V until the gel front reaches the end of the gel.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.
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