![]() ![]() 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.Primary antibody (specific for protein of interest).The advantage is that standard immunodetection may require less optimization for new sample types. The drawbacks of this method are the need for blocking and the total time requirement of over 4 hours. The unoccupied membrane binding sites on the wet blot are blocked with optimized reagents. (If the membrane was dried after transfer, thoroughly wet the blot for 1 minute in methanol if using PVDF or Milli-Q water if using nitrocellulose before proceeding to immunodetection.) Standard immunodetection is performed on blotted proteins directly after electrotransfer. Standard immunodetection, however, offers higher sensitivity and requires less optimization for new sample types. The rapid immunodetection method works well to quickly visualize higher abundance proteins. Rapid immunodetection eliminates the blocking step and reduces the time necessary for the washing and incubation steps. Incubating the membrane with a substrate that reacts with the conjugated secondary antibody to reveal the location of the protein.Washing to remove any unbound secondary antibody.Incubating the membrane with a conjugated secondary antibody, which binds the first antibody.Washing to remove any unbound primary antibody.Incubating the membrane with primary antibody, which binds to the protein of interest.Blocking unoccupied membrane sites to prevent nonspecific binding of antibodies.Standard Immunodetection Methods Include the Following Steps: There are two types of protocols for immunodetection: Standard and rapid. New protocol saves time and delivers consistency. ![]()
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