WesternBlottingProtocols

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Protocol

Standard vs. Rapid Immunodetection Procedures

There are two types of protocols for immunodetection: Standard and rapid.

Standard vs. Rapid Immunodetection

Standard Immunodetection Methods Include the Following Steps:

1. Blocking unoccupied membrane sites to prevent nonspecific binding of antibodies

2. Incubating the membrane with primary antibody, which binds to the protein of interest

3. Washing to remove any unbound primary antibody

4. Incubating the membrane with a conjugated secondary antibody, which binds the first antibody

5. Washing to remove any unbound secondary antibody

6. Incubating the membrane with a substrate that reacts with the conjugated secondary antibody to reveal the location of the protein

Rapid immunodetection eliminates the blocking step and reduces the time necessary for the washing and incubation steps. The rapid immunodetection method works well to quickly visualize higher abundance proteins. Standard immunodetection, however, offers higher sensitivity and requires less optimization for new sample types.

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Standard Immunodetection Method

Application

Standard immunodetection is performed on blotted proteins directly after electrotransfer. (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.)

The unoccupied membrane binding sites on the wet blot are blocked with optimized reagents. The drawbacks of this method are the need for blocking and the total time requirement of over 4 hours. The advantage is that standard immunodetection may require less optimization for new sample types.

Required Solutions

• Primary antibody (specific for protein of interest)

• Secondary antibody (specific for primary antibody), labeled with alkaline phosphatase or horseradish peroxidase.

• Substrate appropriate to the enzyme conjugate (HRP or AP).

• 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.

• Blocking solutions: bovine serum albumin (BSA), 0.2%–5% (w/v), Tween-20,(0.05–0.1%), non fat dry milk (0.5–5%), Casein(1%).

• Milli-Q water

Required Equipment

• Shallow trays, large enough to hold blot

• Glass plates

• Plastic wrap (e.g., Saran? film), freezer bag, or sheet protector

• Autoradiography film and cassette

• Dark room

• Autoradiography film processing equipment

Set Up

1. Dilute the primary antibody in the blocking solution to the desired working concentration.

2. Dilute the secondary antibody in the blocking solution to the desired working concentration.

Note: Enough solution should be prepared to allow for 0.1 mL of antibody solution (primary and secondary) per cm2 of membrane.

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Protocol

1. Perform SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and transfer the protein to the membrane (electroblotting)

2. Wash the membrane twice with distilled water. If desired, stain the membrane with Ponceau Red solution for 5 minutes to visualize protein bands. (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. The Ponceau Red stain will be washed off the membrane during the blocking step. Note: Do not let the blot dry out at any step through development, as this will cause an increase in background staining.

3. Block the blotted membrane in freshly prepared TBS and/or PBS containing nonfat dry milk (3–5% ) (see note on blocking) for 30–60 minutes at room temperature with constant agitation. A maximum blocking time of 2 hours at room temperature should not be exceeded since staining artifacts will appear. If longer blocking times are required, the membrane should be kept at 4°C.

   Note on blocking: Soak the blotted membrane in freshly prepared blocking reagent, PBS/3% nonfat dry milk (15gms nonfat milk in 500mLs PBS) for 30 minutes to 2 hours at room temperature with constant agitation. Membranes may also be blocked with PBS/3% nonfat dry milk overnight at 4°C. Generally, phospho-antibodies require blocking reagents diluted in TBS rather than PBS. If previous Western blots had high backgrounds, try a different blocking buffer. Oter blocking reagents which can be used include a) 3–5% Nonfat dry milk/0.05–0.1%Tween, b) Tween-20 (0.05–0.2%) in PBS or TBS and c) 0.2–5% BSA fraction V in PBS or TBS. Generally, maximum blocking time should not exceed 2 hours at room temperature or proteins can be exchanged from the membrane.

4. Dilute the primary antibody to the recommended concentration/dilution in fresh blocking solution (TBS and/or PBS/3% nonfat dry milk). Incubate the membrane in the primary antibody solution for 1 to 2 hours at room temperature or overnight at 4°C with agitation.

5. Wash the membrane three times for 3 to 5 minutes each with either water or TBS and/or PBS containing 0.05% Tween 20.

6. Incubate the membrane in the secondary antibody reagent of choice for 30 minutes to 1 hour at room temperature or overnight at 4°C with agitation. For a mouse monoclonal antibody, a goat anti-mouse HRP-conjugated antibody is recommended, for a rabbit polyclonal antibody, a goat anti-rabbit HRP-conjugated antibody is advisable.

7. Wash the membrane five times for 3 to 5 minutes each time with either water or TBS and/or PBS containing 0.05% Tween 20.

   Note:Tween-20 detergent has the potential to strip low affinity primary antibodies, and therefore the membrane is briefly washed to improve the background. If the membrane has been washed with water, a final wash step for 3 to 5 minutes in TBS and/or PBS containing 0.05% Tween 20 should follow.

8. Perform the detection of proteins using detection system of choice, (e.g., enhanced chemiluminescence (ECL))

   Note: If Tween-20 is not exhaustively removed by washing the membrane with either water, TBS and/or PBS, the Tween-20 could react with ECL reagent, and there may be an increase in overall membrane staining resulting in a black film.

   .

Western Blotting

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Detection Substrates

Chromogenic Detection

1. Prepare the substrate according to manufacturer’s instructions.

2. Place the blot in a clean container and add substrate to completely cover the surface of the membrane. Incubate for 10 minutes with mild agitation or until signal reaches desired contrast.

3. Rinse the blot with Milli-Q water to stop the reaction.

4. Store the blot out of direct light to minimize fading. Blot may be stored dry.

Chemiluminescent Detection

1. Follow manufacturer’s instructions.

2. Prepare the substrate according to manufacturer’s instructions.

3. Place the blot in a container and add substrate to completely cover the membrane. Incubate for 1 minute.

4. Drain excess substrate.

   Note: A cut-to-size sheet protector or a freezer bag can also be used.

5. Gently smooth out any air bubbles. In a dark room, place the wrapped membrane in a film cassette.

6. Place a sheet of autoradiography film on top and close the cassette.

7. Expose film. Multiple exposures of 15 seconds to 30 minutes should be done to determine the optimum exposure time; 1 to 5 minutes is common.

Fluorescent Detection

REQUIRED EQUIPMENT

• Proteins blotted onto Immobilon-FL transfer membrane and probed with antibodies

• Mylar? wrap

• Fluorescent imaging equipment

The following is a general protocol for fluorescent immunodetection. For optimal results, refer to manufacturer’s protocol provided with the reagents.

Note: If using chemifluorescent reagents, follow reagent manufacturer’s directions.

1. Prepare the substrate according to manufacturer’s instructions.

2. Place the blot in diluted fluorescent dye-labeled secondary antibody solution and incubate for 1 hour with gentle agitation.

3. Wash the blot with wash buffer 3–5 times for 5 minutes each.

4. Place the blot onto a piece of clean filter paper to dry.

5. If using a wrap, use Mylar. Do not use Saran? wrap because it quenches the fluorescence.

6. Image the blot using an appropriate fluorescence scanner.

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Rapid Immunodetection Method

Application

Rapid immunodetection takes advantage of the fact that antibodies cannot bind to the hydrophobic (non-wetted) surface of the Immobilon-P transfer membrane, but will bind to a protein immobilized on the membrane. Rapid immunodetection is compatible with both chromogenic and chemiluminescent substrates. The major advantage of rapid immunodetection is that blocking is not required, saving time and eliminating the risks involved (Mansfield, 1994). The method can be used on both dry and wet Immobilon PVDF membranes, enabling the analysis in about 2 hours, as opposed to over 4 hours for the standard method.

Important: When using a dry membrane, the blot must be thoroughly dry before beginning rapid immunodetection (refer to Membrane Drying Methods).