TITLE: Detailed Western Blotting (Immunoblotting) Protocol
AUTHORS: Rasheed     Sule, Gabriela  Rivera, Aldrin  V Gomes

[DESCRIPTION]
Western Blotting, which is probably better referred to as immunoblotting, is one of the most commonly used biological methods worldwide. This technique is capable of detecting an individual protein from a complex mixture of proteins extracted from cells or tissues. The major steps in the Western Blotting workflow are 1) The separation of proteins based on size, 2) The transfer of separated protein to a suitable stable support, 3) Interaction between the target protein and appropriate primary antibodies. In many cases, a secondary antibody that interacts with the primary antibody is used, and 4) Visualization of the target protein using enhanced chemiluminescence (ECL), fluorescence, or colorimetric methods. A detailed protocol to help users get the most out of their Western Blots is presented.

Goal: To help Western Blotting users learn what prevents them from having perfect Western Blots

The most common result of your experiment:

A good Western blot.

Another possible result:

No signal detected or weak signal

Most likely reasons:

1. Antibody was not suitable (poor quality antibody).
2. Insufficent protein loaded on the gel for the amount of antibody used (or antibody too dluted).
3. Transfer efficiency from gel to membrane was poor. 
2. ECL reagent was expired or contaminated.
5. Incorrect secondary antibody used.
6. Blocking agent concentration was too high.

For more reasons read  WESTERN BLOTTING TIPS AND TROUBLESHOOTING GUIDE

[BEFORE_START]
Although the western blotting procedure is straightforward and well documented, problems can arise, leading to unexpected results.
A significant cause of problems is poor quality samples. For example, if samples were not stored properly before denaturation in sample buffer, many proteins may be proteolyzed and give artifactual results. Invest the time to ensure your samples are well prepared because, even if you do every other part of Western blotting perfectly, poor quality samples will result in inaccurate Western blots.

[GUIDELINES]
The number of companies selling products for Western Blotting continues to increase. However, no reports of any company selling harmful Western blotting products has been reported. As such, feel free to use products from any company, except antibodies. We recommend searching for antibody companies and reviews as well as for the specific antibody and reviews as some companies' antibodies have been reported to be substandard. The most common cause of Western blotting problems is due to the primary antibody used.

[STEPS]
SECTION: Sample preparation
1. Sample preparation can be simple or complex, depending on the source of the sample and the location of the target protein. It is recommended that you consult a dedicated article with procedures for optimal sample preparation.
This protocol assumes you have already prepared your sample and are ready to begin mixing with gel electrophoresis sample buffer.
SECTION: Loading and Running the Gel
2. Loading the sample is important as errors in loading will result in inaccurate results. 
Prepare the electrophoresis tank by placing the gel inside the tank and fill with SDS-PAGE running buffer. Use the same pipette for loading all the wells and if the amount loaded should be the same then ensure that the level of the sample in each lane is the same by eye. 

SECTION: Loading and Running the Gel
2.1. Load a molecular weight marker in appropriate wells of the SDS-PAGE gel, such as lane 1. Bio-Rad Precision Plus protein Standard Catalog #161-0374 (Bio-Rad Laboratories) works well. 

 


SECTION: Transferring the protein from the gel to the membrane
None. Antibody staining
Place the membrane in a blotting container and add enough blocking solution (3% Milk w/ 1/3X TBST, or 1% BSA) to cover the entire membrane surface. Pour solutions into the corner of the blotting container and not directly onto the membrane so that the proteins on the membranes are not disturbed. Incubate the membrane in a shaker for 1 h. Pour out the blocking solution and wash three times with TBST for 3 min each in a shaker.
SECTION: Loading and Running the Gel
2.3. Run the gel for 1–2 hr at 120 V until the dye front reaches the agarose layer of the gel. Depending on the gel system you can run at higher voltages (consult your equipment manufacturer's guide). 
When the dye front (caused by the bromophenol blue in the Laemmli buffer) reaches near the bottom or at the bottom of the gel,  remove the gel from the glass or plastic plates. It is recommended that the wells be removed with a razor blade or similar object as this helps reduce your chance of having bubbles during transfer and makes handing of the gel easier. 
Make a small cut at the top left side of the gel for orientation.

 

SECTION: Transferring the protein from the gel to the membrane
3. 
The membrane can be either nitrocellulose or polyvinylidene fluoride (PVDF). There are a few differences between the two membranes:
 
 
     Nitrocellulose      PVDF        Less Expensive      More Expensive        Fast Protein Binding      Higher Sensitivity Protein Binding        Does Not Require Pre-Wetting      Requires Pre-Wetting with Methanol        Weaker Than PVDF      Stronger Than Nitrocellulose        Not Recommended for Stripping and   Reprobing      Recommended for Stripping and   Reprobing               Good for Hydrophobic Proteins               Requires Re-wetting with Methanol   Throughout Use   

SECTION: Loading and Running the Gel
2.4. The gel percentage required is dependent on the size of your protein of interest:
 
 
     Protein   size      Gel   percentage        4–40   kDa      20%[GR1]         12–45   kDa      15%[GR2]         10–70   kDa      12.5%        15–100   kDa      10%[GR3]         25–100   kDa      8%        60-210   kDa      5%[GR4]    

Gradient gels can also be used.
 
     Protein   size      Gel   percentage        5-200   kDa      4-12   gradient        4-200   kDa      4-20   gradient        3.5-110   kDa      10-20   gradient   
 

 


 

 
 
SECTION: Transferring the protein from the gel to the membrane
3.1. Activate PVDF with methanol for 1 min and rinse with transfer buffer before preparing the stack. Transfer of proteins to the membrane can be checked using Ponceau S staining before the blocking step.
SECTION: Transferring the protein from the gel to the membrane
3.4. Prepare the stack as follows:
  

SECTION: Transferring the protein from the gel to the membrane
3.2. If using a wet or tank transfer, soak one stack of filter paper in transfer buffer and place it on the sponge. Wet the nitrocellulose membrane with transfer buffer and lay it on top of the filter paper stack. Allow at least 2 min for the filter paper and membrane to soak in the transfer buffer. Place the gel on top of the membrane, followed by another stack of filter paper soaked in transfer buffer. 
Firmly roll out any air bubbles with a roller (such as a clean pencil). Use your fingers to apply mild pressure on the whole transfer stack while closing the cassette to ensure no additional air bubbles form. Once the stack has been completed, place the stack into the tank and fill the tank with transfer buffer. The tank should be run between 0.1 to 1 amps or 5 to 30 V for an hour or overnight, depending on the amp or voltage. 


 

 

SECTION: Transferring the protein from the gel to the membrane
3.3. If using the Trans-blot Turbo (fast transfer), soak one stack of filter paper in transfer buffer and place it in the Trans-Blot Turbo Transfer System cassette. Wet the nitrocellulose membrane with transfer buffer and lay it on the filter paper stack. Allow at least 2 min for the filter paper and membrane to soak in the transfer buffer. Place the gel on the membrane, followed by another stack of filter paper soaked in transfer buffer. Firmly roll out the air bubbles with a roller. Use your fingers to apply mild pressure on the whole transfer stack while closing the cassette lid to ensure no additional air bubbles form. Wipe the outside of the cassette with tissue paper and place it inside the Trans-Blot Turbo Transfer System machine. Run with the appropriate setting. We use midi gels so our transfer uses the Turbo midi setting of 7 min to transfer the proteins from the gel to the membrane.
SECTION: Transferring the protein from the gel to the membrane
3.5. Take the membrane from the cassette and image it while still wet in the ChemiDoc MP Imaging System. If using  Stain-Free gels, then use the Stain-Free membrane setting and optimized exposure time. There is no need to activate the membrane again. Adjust the exposure time as needed to avoid overexposed images. If necessary, the membrane can be cut into smaller sizes at this stage.  If using normal gels use Ponceau S to stain the membranes.

 

SECTION: Transferring the protein from the gel to the membrane
3.6. Dry the membrane on top of a piece of tissue paper. Allowing the membrane to dry before proceeding to immunostaining has been suggested to help the proteins adhere better to the membrane.

 

SECTION: Transferring the protein from the gel to the membrane
None. Washing
Wash the membrane in three washes of 1/3X TBST, 3 min each.
SECTION: Transferring the protein from the gel to the membrane
None. Place the membrane in a blotting container and add enough blocking solution (3% Milk w/ 1/3X TBST, or 1% BSA) to cover the entire membrane surface. Pour solutions into the corner of the blotting container and not directly onto the membrane so that the proteins on the membranes are not disturbed. Incubate the membrane in a shaker for 1 h. Pour out the blocking solution and wash three times with TBST for 3 min each in a shaker.
SECTION: Transferring the protein from the gel to the membrane
None. Incubate the membrane with appropriate dilutions of the primary antibody in blocking buffer (1% non-fat milk in TBST, or 3 % BSA in TBST). We recommend overnight incubation at 4°C, followed by incubation with shaking for 1 h at room temperature. 
SECTION: Transferring the protein from the gel to the membrane
None. Secondary antibody
Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer (1% Milk w/ 1/3X TBST) at room temperature for 1 h.
SECTION: Transferring the protein from the gel to the membrane
None. Washing
Wash the membrane in three washes of 1/3X TBST, 3 min each.
SECTION: Transferring the protein from the gel to the membrane
None. Detection
SECTION: Transferring the protein from the gel to the membrane
None. 
SECTION: Transferring the protein from the gel to the membrane
None. For signal development, follow the kit manufacturer’s recommendations. Remove excess reagent and cover the membrane in transparent plastic wrap.
SECTION: Transferring the protein from the gel to the membrane
None. In a 1.5 mL centrifuge tube, mix together equal parts of Clarity western peroxide agent and Clarity western luminol/enhancer reagent. This creates the substrate for enhanced chemiluminescence (ECL). Pipette enough ECL substrate to evenly cover the surface of the membrane where the proteins are attached. Incubate the ECL covered membrane in a dark condition for 2 min before removing the excess ECL reagent.
SECTION: Transferring the protein from the gel to the membrane
None. Pick up the membrane from one side with a pair of Forceps and drain away the excess ECL by gently touching the edge of the membrane to a delicate wiper paper. Arrange the membrane on the imaging surface, taking care not to create bubbles on the surface.
SECTION: Sample preparation
1.1. Remove a small volume of sample to perform a protein quantification assay. Determine the protein concentration for each cell sample. Use a protein quantification appropriate method that is compatible with the reagents in your lysis/homogenization buffer.

 

 

SECTION: Sample preparation
1.2. Determine how much protein to load in each well and how many wells you will load. If you are going to load many wells on different gels over several weeks then prepare them at the same time. Once you know how much sample you need, dilute your sample appropriately or use it as is and add an equal volume of 2X Laemmli sample buffer to get the final concentration of your protein at a concentration you want.  We recommend reducing and denaturing the samples unless the online antibody datasheet indicates that non-reducing and non-denaturing conditions should be used.

 

SECTION: Sample preparation
1.3. To reduce and denature your samples, heat each cell lysate in sample buffer at 75°C for 5 mins. Allow samples to cool and then use or aliquot and store at -20°C for future use (good for at least three months).  For longer storage store at -80°C. 

 


 

 

 

SECTION: Transferring the protein from the gel to the membrane
None. 
SECTION: Primary Antibody Staining
5. The most critical part of any Western blot is the primary antibody. 
SECTION: Primary Antibody Staining
5.1. Incubate the membrane with appropriate dilutions of primary antibody in blocking buffer (1% Milk in TBST, or 3 % BSA in TBST). We recommend overnight incubation at 4°C, or at least 4 hours incubation at room temperature. 


 

 

 

SECTION: Washing
6. Although it may seem like a trivial step, washing is important for improving the signal to noise (background) so that quantification is more accurate. 
SECTION: Washing
6.1. Wash the membrane in three washes of TBST, 3 min each.

 

 

SECTION: Secondary Antibody
7. The choice of companies to purchase secondary antibodies is huge. Some companies have well established secondary antibodies that are respected and work well. We have used secondary antibodies from Cell Signaling, Bio-Rad, Sigma, and Abcam and they all worked well.
SECTION: Secondary Antibody
7.1. Incubate the membrane with the recommended dilution of conjugated secondary antibody in blocking buffer (1% Milk in TBST) at room temperature for 1 hr. For chemiluminescence, the secondary antibody is conjugated with horseradish peroxidase (HRP).
SECTION: Washing Part II
8. Time to remove excess secondary antibodies.
SECTION: Washing Part II
8.1. Wash the membrane in three washes of TBST, 3 min each.
SECTION: Detection
9. The choice of signal development reagents is expanding yearly. Most commercial preparation work well. Of 21 different commercial products we have tried, only one was disappointing.
SECTION: Detection
9.1. For signal development, follow the kit manufacturer’s recommendations the first time you use their reagents. 
SECTION: Detection
9.2. Because of price and sensitivity, we utilize Clarity enhanced chemiluminescence (ECL) reagent. The amount of ECL reagent depends on the size of the blot. For a 3 cm x 4 cm membrane strip, we use 1 ml of ECL reagent. In a 1.5 mL centrifuge tube, mix equal parts of Clarity western peroxide agent (0.5 mL) and Clarity western luminol/enhancer reagent (0.5 mL). This creates the substrate for chemiluminescence. Pipette enough ECL substrate to evenly cover the surface of the membrane where the proteins are attached. Incubate the ECL covered membrane in a dark condition for 2 min before removing the excess ECL reagent.


 

SECTION: Imaging
9.3. Pick up the membrane from one side with a pair of blunt forceps and drain the excess ECL reagent by gently touching the edge of the membrane to a delicate tissue paper. Arrange the membrane on the imaging surface, taking care not to create bubbles on the surface.
SECTION: Imaging
9.4. Image the blot on your machine of choice. We utilize the Bio-Rad ChemiDoc MP Imaging system with the Chemi Hi Sensitivity setting and 10 seconds of imaging time initially. If the image is saturated (red bands) then we use the Chemi Hi-Resolution setting with the optimized exposure time. If the initial 10 s image was not visible, we do a 60 s high sensitivity image. 
The exposure time can be adjusted as needed to obtain the best image that contains no overexposed (saturated) bands. 
One of the advantages of the ChemiDoc MP is that we are able to take a multichannel image with the stain-free and either the Chemi Hi Sensitivity or Hi-Resolution setting to get an image of the chemiluminescent protein bands of interest overlaid with the stain-free prestained protein markers. 
Although the other proteins on the membrane will also show up with the stain-free image, the chemiluminescent signal shows up on a different channel. Verify the molecular weight of the band of interest based on the multichannel image.
 

SECTION: Imaging
10. After you get your great looking blots you may want to quantify your results. 
A major concern of western blot quantification is the difficulty associated with determining saturated signals. This is especially problematic when using film. It is recommended that a digital imager be used (many universities now have digital imagers so try to find a lab that has one). Please read the manufacturer’s recommendation on how to use the specific imager you are using as well as do a google search for problems with quantification using the software that you will use to quantify. Unfortunately, after getting great blots some researchers do inaccurate quantification of their results. Invest your time to learn how to quantify the signals accurately.
SECTION: Loading and Running the Gel
2.2. We typically load 8-15 μg of total protein for cell lysates or tissue homogenates or 10–100 ng for purified protein. Larger amounts of protein can result in non-linearity when housekeeping proteins are used as normalization standards. 
SECTION: Relax
11. Sit back and enjoy your Western blotting results.


 

SECTION: Blocking
4. Blocking is an important step in the Western blotting process. Blocking prevents the non-specific binding of antibodies to the membrane.

SECTION: Blocking
4.1. Place the membrane in a blotting container and add enough blocking solution (3% Milk in TBST, or 5% BSA in TBST) to cover the entire membrane surface. Pour solutions into the corner of the blotting container and not directly onto the membrane so that the proteins on the membranes are not disturbed. Incubate the membrane on a shaker for 1 h. Pour out the blocking solution and rinse one time with TBST for 10-20 seconds to remove excess milk.