08x_IMMUNoDOT BLOT_111209

08x_IMMUNoDOT BLOT_111209 - ImmunoDot Blot (Immunobloting)...

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ImmunoDot Blot ( Immunobloting ) LABORATORY EXERCISE #8 Rev: 3/10/10 Immunobloting Antibody binding to antigens was evaluated by immunoblotting. Ten µl of 1:5 serial dilutions of antigens (100 µg/ml in TBS) dotted and air-dried onto nitrocellulose membrane (Protran BA85; Schleicher & Schull, Keene NH). The membrane was then treated for 30 min with assay buffer (TBS containing 1% skimmed milk) room temperature, followed by three washing steps with TBS. Antisera specific for the target antigens (………, diluted 1:1000 in assay buffer) were added, blots incubated at 37 o C for 90 min, then washed three times with TBS. Enzyme-conjugate (Protein A- HRP; Zymed Laboratories, S. San Francisco CA ;diluted 1:1000 in assay buffer) was added to the blot and incubated at 37 o C for 45 min, then washed. A final wash with TBS was carried out before developing the blot using a chromatogenic substrate ( HRP  color developing reagent  prepared according to the manufacturer’s instructions; Biorad,  Hercules CA).   After developing the blot was washed with TBS and allowed to dry at room temperature. Immunodetection – Enzyme Immunoassay (EIA) Reaction catalyzed by horseradish peroxidase (HRP) using BioRad HRP color developing reagent (4-chloro-1-napthol substrate) 1
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Procedure :
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08x_IMMUNoDOT BLOT_111209 - ImmunoDot Blot (Immunobloting)...

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