Lab4 - Western Transfer - Immunoblots


List of Supplies

Run PCR products on gel
1. place gel in gel box and fill with 1x TAE buffer (to fully cover wells)
2. remove combs from wells
3. load 7uL 100bp ladder in far left lane
4. load 25uL of your PCR sample into the gel (retain the remaining vol at -20ºC)
5. run gel at ~ 100V for ~ 1hr
6. visualize the gel on the UV transilluminator
Run Protein Gel [as in Lab 2]
Principles of Western Blotting:
Protein Transfer:
After the protein components have been sufficiently separated by electrophoresis, they can be transferred to a nitrocellulose membrane. The transfer process uses the same principle as SDS-PAGE – this time the electric current is applied at 90 degrees to the gel and the proteins migrate out of the gel onto the membrane. The membrane used for our lab is a positively charged nitrocellulose membrane. Remember from the sample preparation for SDS-PAGE, we've treated the proteins with SDS to impart an overall negative charge to these molecules. During transfer, the proteins migrate through the electric field, out of the gel and onto the membrane. The negative charge of the proteins allows them to efficiently bind to the positively charged membrane. However, nitrocellulose membranse will bind virtually any proteins, regardless of charge. The charge simply ensures a stronger bond. Thus, it is extremely important to handle nitrocellulose membranes with clean gloves and clean utensils to minimize extraneous proteins from being bound to the membrane.
Western Blot Procedure:
• Blocking
The membrane is blocked in order to reduce non-specific protein interactions between the membrane and the antibody. This is achieved by placing the membrane in a solution of non-specific proteins (usually BSA or non-fat milk). The proteins in the blocking solution coat the remaining areas of the membrane where no protein is bound from the transfer. The reason this is necessary is described in the next step.
• Primary Antibody
The first antibody to be applied (specific for protein of interest) is incubated with the membrane. The primary antibody is specific for the protein of interest (in this case HSP70), and, at appropriate concentrations, should not bind any of the other proteins on the membrane. Remember, antibodies are proteins, too. If we had not blocked the membrane, the antibody would end up binding to both the membrane and your target protein. This would result in extremely high background (signals not related to the intended target protein(s)) and would use up a significant amount of the available antibody, making the interpretion of results difficult, if not impossible.
• Secondary Antibody
After rinsing the membrane to remove unbound primary antibody, a secondary antibody is incubated with the membrane. It binds to a species-specific portion of the primary antibody. Due to its targeting properties, the secondary antibody tends to be referred to as "anti-mouse," "anti-goat," etc., depending on the animal species that the primary antibody was created in. This secondary antibody is typically linked to an enzyme that allows for visual identification. In our case, the antibody is linked to an alkaline phosphatase (AP).
• Developing
The unbound secondary antibodies are washed away, and the enzyme substrate is incubated with the membrane so that the positions of membrane-bound secondary antibodies will either change color or emit light. Bands corresponding to the detected protein of interest can be visualized. Band densities in different lanes can be compared providing information on relative abundance of the protein of interest. The kit we will be using for visualization (Invitrogen's WesternBreeze Chromogenic Kit) utilizes an enzymatic reaction that creates a dark purple precipitate that can be seen with the naked eye.

The chromogenic system emplyed in the WesternBreeze Chromogenic Kit is the combination of BCIP (5-Bromo-4-Chlo ro-3'-Indolypho sphate p-Toluidine Salt) and NBT (Nitro-Blue Tetrazolium Chloride). Together they yield an intense, insoluble black-purple precipitate when reacted with Alkaline Phosphatase. The NBT/BCIP reaction is illustrated in the figure below. This reaction proceeds at a steady rate, allowing accurate control of the relative sensitivity and control of the development of the reaction.

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Transfer Proteins to membrane

1. Cool the transfer buffer to 4°C.
2. Soak the filter paper, membrane and gel in Transfer Buffer for 15 minutes.
3. Assemble the blotting sandwich in a semi-dry blotting apparatus as follows:
• Anode (+++)
• Filter paper
• Nitrocellulose Membrane
• Gel
• Filter paper
• Cathode (– – –)
4. Transfer the blot for 30 minutes at 20V.
5. Remove the gel from the sandwich and rinse with transfer buffer.
6. Use a cotton swab to remove any adhering gel from the membrane.

Western Blotting Protocol
Western Breeze Manufacturer's Protocol
General Guidelines
• Avoid touching the working surface of the membrane, even with gloves.
• Work quickly when changing solutions as membranes dry quickly. If the membrane dries, re-wet the membrane with methanol and rinse with water before proceeding.
• Add solutions to the trays slowly, at the membrane edge, to avoid bubbles forming under the membrane. Decant from the same corner of the dish to ensure complete removal of previous solutions.

1. Prepare 20 mL of Blocking Solution
Ultra filtered Water 14 ml
Blocker/Diluent (Part A) 4 ml
Blocker/Diluent (Part B) 2 ml
Total Volume 20 ml

2. Place the membrane in 10 ml of the appropriate Blocking Solution in a covered, plastic dish provided in the kit. Incubate for 30 minutes on a rotary shaker set at 1 revolution/sec.

3. Decant the Blocking Solution.

4. Rinse the membrane with 20 ml of water for 5 minutes, then decant. Repeat once.

5. Prepare 10 mL of Primary Antibody Solution (1:3000 dilution)
Blocking Solution 10 ml
HSP 70 antibody 3.3 µl
Total Volume 10 ml

6. Incubate the membrane with 10 ml of Primary Antibody Solution for OVERNIGHT



NEXT DAY

Decant Primary Ab, saved at 4C.

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