![]() ![]() If previous Western blots had high backgrounds, try a different blocking buffer. Generally, phospho-antibodies require blocking reagents diluted in TBS (Cat No. Membranes may also be blocked with PBS/3% nonfat dry milk overnight at 4☌. Note on blocking: Soak the blotted membrane in freshly prepared blocking reagent, PBS/3% nonfat dry milk (15gms nonfat milk in 500mLs PBS (PBS Tablets 524650-1EA) for 30 minutes to 2 hours at room temperature with constant agitation. ![]() If longer blocking times are required, the membrane should be kept at 4☌. A maximum blocking time of 2 hours at room temperature should not be exceeded since staining artifacts will appear. 524750-1EA) and/or PBS (PBS Tablets 524650-1EA) containing nonfat dry milk (3–5% ) (see note on blocking) for 30–60 minutes at room temperature with constant agitation. Note: Do not let the blot dry out at any step through development, as this will cause an increase in background staining.īlock the blotted membrane in freshly prepared TBS (Cat No. The Ponceau Red stain will be washed off the membrane during the blocking step. (Stock solution: 2% Ponceau S in 30% trichloroacetic acid and 30% sulfosalicylic acid dilute 1:10 for use.) Rinse the membrane in water until protein bands are distinct and mark the position of the molecular weight markers with a ballpoint pen or pencil. If desired, stain the membrane with Ponceau Red solution for 5 minutes to visualize protein bands. Wash the membrane twice with distilled water (Cat No.Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer the protein to the membrane (electroblotting).Note: Enough solution should be prepared to allow for 0.1 mL of antibody solution (primary and secondary) per cm2 of membrane. Dilute the secondary antibody in the blocking solution to the desired working concentration.Dilute the primary antibody in the blocking solution to the desired working concentration.Autoradiography film processing equipment.Plastic wrap (e.g., Saran™ film), freezer bag, or sheet protector.Shallow trays, large enough to hold blot.Appropriate wash buffer: Phosphate-buffered saline (PBST): 10 mM sodium phosphate, pH 7.2, 0.9% (w/v) NaCl, up to 0.1% Tween-20 detergent, TBST: 10 mM Tris, pH 7.4, 0.9% (w/v) NaCl, up to 0.1% Tween-20.Substrate appropriate to the enzyme conjugate (HRP or AP).Secondary antibody (specific for primary antibody), labeled with alkaline phosphatase or horseradish peroxidase. ![]()
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