Western blot

WESTERN BLOT
	Last Update: December 2006
	This protocol uses SDS/Native PAGE gels

PREPARE SOLUTIONS
1. 10X Running buffer (5 L):	Mix 151 g of Tris base and 720 g of Glycine. Add dH₂O to 5 L and store at 4^oC
2. 1X SDS-running buffer (1 L):	Mix 10 mL of 10% SDS, 100 mL of 10X Running buffer, and 890 mL of dH₂O
3. Transfer buffer (1 L):	Mix 250 mL of 1X SDS-Running buffer, 200 mL of Methanol, and 550 mL of dH₂O
4. 10X Ponceau solution (100 mL):	Mix 2 g of Ponceau-S (2%), 30 g of Trichloroacetic acid (30%), 30 g of Sulfosalicylic acid (30%), and dH₂O to 100 mL
5. Alkaline buffer (1 L):	Mix 12.1 g of Tris base (100 mM), 5.8 g of NaCl (100 mM), and 1 g of MgCl₂ (5 mM). pH to 9.5 with HCl
6. PBS (1 L):	Mix 8 g of NaCl, 0.2 g of KCl, 1.44 g of Na₂HPO₄, 0.24 g of KH₂PO₄, and 800 mL of dH₂O. pH to 7.4 with HCl and add dH₂O to 1 L (Optional: Autoclave)
7. PBST_0.1 (PBS + 0.1% Tween-20):	Mix 1 L of PBS with 1 mL of Polyoxyethylene-sorbitan monolaurate (Tween-20)
8. 100X NBT (5 mL):	Mix 150 mg of NBT (chloride), 3.5 mL of DMF, and 1.5 mL of dH₂O. Store in the dark at 4^oC
9. 100X BCIP (5 mL):	Mix 75 mg of BCIP with 5 mL of DMF. Store in the dark at 4^oC

PROCEDURE
NOTE: the amount of Tween-20 added to PBS should be determined for each application. Some antibodies will work fine with as low as 0.1% Tween-20, but background levels can be reduced and even removed completely with concentrations of up to 1%
1. After running the Acrylamide gel (SDS/Native PAGE) for the appropiate length of time, soak the gel in transfer buffer and set up the Western transfer with one of the following two setups:
1.1 For Semi-dry set up: positive pole -> 2-3 3MM papers (previously soaked in trasnfer buffer) -> Nitrocellulose membrane (Hybond-C) (previously soaked in transfer buffer) -> Acrylamide gel -> 2-3 3MM papers (previously soaked in trasnfer buffer) -> negative pole
1.2 For Wet transfer set up: positive pole -> porous membrane ->1-2 3MM papers (previously soaked in trasnfer buffer) -> Nitrocellulose membrane (Hybond-C) (previously soaked in transfer buffer) -> Acrylamide gel -> 1-2 3MM papers (previously soaked in trasnfer buffer) -> porous membrane -> negative pole
2. Run at constant Amps (Amperage = surface area of nitrocellulose membrane (in cm²) X2). For Homemadegels (SDS/Native PAGE) in a Semi-dry set up = 450 mAmps for 2-3 hours (make sure voltage never goes over 20 V; if it does, lower voltage). For a Wet transfer set up = 30 mAmps overnight (~40 Volts).
3. Disassemble set up and place membranes in water (optional: stain gels (Protein staining) to determine transfer efficiency). Stain with 1X Ponceau (5-15 minutes in a rocker)
4. Take a picture of membrane and mark size markers prior to washing Ponceau away using water (or PBS)
5. Block overnight using PBST + 10% Non-fat milk (or 3-5% BSA)
6. Throw away PBST + 10% Non-fat milk and wash with PBST while preparing a PBST + 1% Non-fat milk solution containing antibody of interest
7. Add PBST + 1% Non-fat milk containing antibody (Azide at <0.1% can be added to this solution to prevent contaminations -the antibody + PBST + 1% Non-fat milk solution can be stored at 4^oC for weeks)
8. Incubate in a rocker for >1 hour (the longer the incubation, the more specific the reaction will be)
9. Save antibody + PBST + 1% Non-fat milk solution and wash 3X (>5 minutes each) with PBST
10. Add PBST + secondary antibody. Incubate in rocker for 1-2 hours
11. Save antibody + PBST and wash 3X (>5 minutes each) with PBST
12. Wash once with Alkaline buffer
13. Develop membranes using Alkaline buffer + 1X NBT + 1X BCIP (look as the signal develops; some antibodies give signals in a matter of seconds and over-exposure can occur, while other may take over an hour to develop)
14. Once the signal has developed to the desired level (note that the signal will develop a little more even after stopping the reaction, so stop it before the desired level otherwise over-exposure will occur), throw away the Alkaline buffer + 1X NBT + 1X BCIP solution and wash quickly with water, followed by a 20mM EDTA solution (EDTA chelates the reaction and stops it)

TIPS - Millipore Product FAQ for Immobilon-P
Question: How can I transfer a range of high and low molecular proteins from my gel onto Immobilon-P without the small proteins being lost? Answer: ramping of the current and optimization of the transfer buffer composition can be used together to give efficient blotting of proteins over a wide range of molecular weight. They may also be used separately to give the maximum transfer efficiency for either small or large proteins. 1. Ramp - To help slow down small proteins, start the transfer at half the normal current (or voltage), and gradually increase the current (or voltage) during the transfer period to the maximum setting recommended. This measure allows the smaller proteins to travel more slowly through the electric field and have more residence time within the membrane. This step increases the chance that binding will occur. As the current is increased the larger proteins start to move out of the gel and onto the membrane as well. 2. Optimize your transfer buffer - When optimizing your transfer buffer, it is important to realize that conditions favoring the transfer and binding of small proteins often have a negative effect on the transfer of large proteins. The converse is also true. The following rules are generally applicable. Most small proteins elute from a gel with little difficulty. To improve their binding to the membrane, SDS should be omitted from the transfer buffer and the methanol concentration should be increased to as high as 40%. For optimal blotting of large proteins, decreasing the methanol concentration to 10% or less and supplementing the transfer buffer with up to 0.05% SDS enhance their solubility and minimize their precipitation within the gel. Thus, they are better able to elute from the gel. Large proteins normally bind to the membrane very readily.