How to Run a Western Blot

This How To Guide Will Cover

• Western Blot Theory Overview
• Sample Preparation
• SDS-PAGE Method
• Transferring Method
• Immunoblotting Method
• Detection Method
• Western Blot Troubleshooting

Western Blot Theory Overview

Antibodies bind to specific sequences of amino acids, known as the epitope. Because amino acid sequences are different from protein to protein, antibodies can recognize specific proteins among a group of many. Therefore, a single protein can be identified in a cell lysate that contains thousands of different proteins and its abundance quantified through Western Blot Analysis. First, proteins are separated from each other based on their size. Second, antibodies are used to detect the protein of interest. Finally, a substrate that reacts with an enzyme is used to view the antibody/protein complex.

Sample Preparation

1. The first step in sample preparation, is isolating the protein from a source. Usually, protein is purified from cells. However, other sources of protein, such as synthetically derived protein, can also be run through a Western blot.

2. Next, the protein concentration is determined. Loading buffer, which contains SDS (sodium dodecyl sulfate), and BME (beta-mercapto ethanol) is added to the protein suspension.

3. The sample is then heated to near-boiling, which denatures the protein and allows the SDS to bind to the protein. SDS carries a negative charge.

SDS-PAGE Method

1. The first step in SDS-PAGE, is placing the sample, containing the protein-SDS compound, in a well on top of the gel. A ladder is usually loaded in one of the wells. Ladders are protein mixtures of known molecular weight, that allow the researcher to determine the molecular weights of the other proteins on the gel.

2. Once the sample(s) and ladder are loaded, a current is run across the gel, with a negative pole on the well end of the gel, and a positive pole on the opposite end of the gel. Because the protein is bound to negatively charged SDS, it is pulled down through the gel to the positive pole. The larger the protein, the slower it moves.

Although running the gel is the last step in the SDS-PAGE method, it is important to make note of several pieces of information:

• Proteins are separated by their size as they run through the gel.
• The lower the concentration of acrylamide in a gel, the easier it becomes for proteins to move through the gel; so all proteins move further under the same conditions.
• Gradient gels are gels where the concentration of acrylamide increases from the top to the bottom

Transferring Method

After the gel is run, it is placed against a membrane, and current is passed across the gel to the membrane, transferring the proteins onto the membrane.

The transferring method detailed in this How To Guide is known as semi-dry, however transferring can also be done using the wet method. The membrane is usually made of PVDF or Nitrocellulose, both of which have advantages and disadvantages that should be throughougly researched prior to use.

Immunoblotting Method

1. The first step in immunoblotting is to wash the membrane and block it with non-specific protein. The non-specific protein binds to the surface of the membrane where protein is not already present. This will prevent antibody from binding to the membrane and giving a non-specific signal.

2. Next, the primary antibody is added to the solution in which the membrane is floating. Remember that the primary antibody recognizes a specific amino-acid sequence of a particular protein.

3. After a wash is conducted to remove unbound primary antibody, secondary antibody is added. Secondary antibody recognizes the primary antibody, and usually is conjugated with an enzyme, such as HRP (Horse Radish Peroxidase).

4. Lastly, another wash is performed to remove unbound secondary antibody. Non-specific binding of both the primary and secondary antibodies can occur, but thorough washing usually minimizes this problem. The amount of time the primary and secondary antibodies are applied, directly affects the specificity and strength of binding.

Detection Method

1. The detection method used is dependent upon the enzyme to which the secondary antibody is conjugated. The most common enzyme used in Western Blotting is HRP, and the substrate used for detection is known as Chemiluminescent substrate. Novus Biologicals' detection substrate is known as NovaLume, and NovaLume-Plus.
2. The below figure displays how the detection substrate (NovaLume) works:

3. Once the substrate has been added, the light being emitted can be detected with film or a photo imager.

4. Notice that the film does not contain information on where the lit bands are located in relation to the membrane. Therefore, it is very important to use a method that allows the film to be aligned with the membrane in the exact same manner as when the film was exposed. The membrane is usually stained after the detection step, so that the protein present can be visualized, and compared to the film.

Western Blot Troubleshooting

Issues with Western Blotting can arise from the sample preparation, running SDS-PAGE gels, as well as transferring, immunoblotting and detecting the gel. Below are some common problems and varriations with these procedures.

Sample Preparation

• Protein can be from almost any source. The important thing is that it is in a solution that is acceptable for Western Blotting (DNA, high salt levels, and lipids can all ruin an experiment, so be sure to remove them).
• Besides using lysis buffer, cold sonication also works as a method for cell lysis.
• Protein can be run through a gel in its native state, using the 'Native Gel' method.

SDS-PAGE

• Loading too much or too little protein can lead to problems.
• Very large proteins (>150kD) may be too large to enter an acrylamide gel, but decreasing the acrylamide concentration may help.
• Very small proteins (<15kD) may run off the gel, and be impossible to visualize, but increasing the acrylamide concentration may help.
• The loading portion of the gel has a lower acrylamide concentration because the intention is to load all proteins of all sizes into the structure of the gel quickly and evenly.
• Malformations in the shape of the wells can lead to malformed bands and poor resolution.
• Gradient gels, in which the resolving portion of the gel changes from a low acrylamide concentration to a high concentration, are useful to see a wide range of protein sizes.
• As the electric current runs through the gel, it will heat the gel and running buffer. Too much heat can destroy your gel, so be sure to keep your whole apparatus cool.
• An improper seal between your gel and the apparatus is very common, so test your assembly before running.

Transferring

• Be sure to research the benefits and drawbacks to PVDF vs. Nitrocellulose prior to conducting your Western Blot. One such variance between these two membrane options is that 100% Methanol will dissolve nitrocellulose, but is a good wetting choice for PVDF.
• Transfer membranes are intended to grab protein, so do not touch the membranes with your bare hands or to any unclean surfaces.
• The current must pass evenly through all the layers of the transfer, so any bubbles or irregularities must be removed in each layer.
• No transfer is perfect, so protein will be left behind in your gel, and some protein will pass all the way through the membrane. Larger proteins are more difficult to transfer, while smaller proteins may transfer too easily.
• Reversing the order of the gel and membrane will mean that no transfer occurs, and your proteins will be lost into the blot paper, so be sure your gel is on the negative side of the transfer sandwich.

Immunoblotting

• Blocking and washing are important steps that decrease noise and improve specificity. Blocking with BSA instead of NFDM (non-fat dry milk) is just as acceptable, and necessary if the proteins of interest may also be present in NFDM.
• Antibodies are proteins, so they are susceptible to damage from heat and freeze/thaw cycles, as well as denaturing agents. Read the datasheet carefully for storage instructions.

Detection

• Chemiluminescent substrates, including NovaLume, come in two bottles because once the two reagents are mixed, they have a very limited shelf life. The mixed reagent should be added to your membrane as quickly as possible.
• Cutting the corners of your membrane and film to line-up exactly is one good method to make sure you can line them up again after processing.
• Dirty/used photochemicals will increase your background noise.
• The length of time the light signal from your enzyme/substrate reaction lasts varies, but is usually less than 1 hour, and decreases significantly within 10 minutes.
• If you do not see anything on your film right after development, try exposing your film for much longer to the membrane. Likewise, if your signal is too strong, decrease the length of time you expose the film to the membrane.
• If increased exposures or the use of a photo imager still give no results, try increasing your primary antibody concentration, or length of application. If you still do not obtain results, the use of positive control will confirm if the antibody is working.
• If your results show detection of a protein at an unexpected molecular weight, check the protein for splice variants, glycosylation, phosphorylation, dimers, etc. Also, check the antigen sequence used to create the primary antibody for homology with other proteins.
• It is not uncommon to see other bands on your western blot. As long as they do not interfere with your band of interest, there should not be a problem. The use of negative and positive controls, as well as loading controls can help determine if the results you are getting are valid and useful.