Sample preparation for western blot

Before setting up a western blot experiment, inform yourself about the characteristics of the protein of interest. Those include: 

• Size 
• Cellular localization 
• Solubility (membrane integrated vs soluble) 
• Modifications (protease processing, post-translational modifications like glycosylation) 
• Known isoforms 
• Protein abundance 

Depending on those specific characteristics, you might need to set up a suitable strategy to be able to detect the target protein using western blotting. 

The preparation of your sample for western blot involves four main steps: 

1. Sample isolation – Fractions, organelles, cells, or tissue must be individually isolated from a certain source of interest. It's essential to consider additional requirements based on the source type. For adherent cells, a simple trypsin treatment may suffice, while uneven tissues may require the removal of unwanted components, such as fat or collagen. Removing these unwanted components may be necessary to enhance the quality of the experiment. 
2. Lysis – The organelles, cells or tissue samples are treated with buffers and mechanically agitated to release proteins. Selecting a suitable lysis strategy is critical for obtaining good-quality results. In some cases, samples like cell culture supernatant may not require lysis, but protein abundance might be low, making sample loading challenging. In such scenarios, protein precipitation techniques may be necessary, like TCA precipitation or protein concentration, using specific spin columns.  
3. Measuring protein concentration – this step is crucial to ensure the loading of sufficient protein and equal amounts of the sample. Keep in mind that some protein concentration measurement techniques may not be compatible with detergent in the lysis buffer or buffer constituents like EGTA. Checking compatibility beforehand helps in selecting the appropriate method for protein determination.  
4. Reducing and denaturing – Samples are also usually treated with agents to disrupt higher-level protein structures (secondary and tertiary structures) and linearize the proteins, ensuring protein separation exclusively by size. The sample buffer often contains DTT or beta-mercaptoethanol to reduce disulfide bonds and SDS to form denatured protein micelles coated with SDS molecules. Additionally, the sample is boiled, or at least heated, for a short time.  

Selecting a specific lysis buffer is vital for targeting a protein located in a particular compartment. The components of the suitable lysis buffer should ensure effective solubilization and detection of the target protein in the sample. For more detailed information and recipes for lysis buffers, refer to Buffers and stock solutions for western blot. 

Enriching a specific organellar fraction, such as mitochondria, may be necessary if working with low-abundance proteins or difficult target proteins with high background. This approach, called cellular fractionation, improves the signal-to-noise ratio by excluding other proteins and reducing sample diversity. Cellular fractionation, typically achieved through a differential centrifugation process, may involve using sugar-based materials for density gradient centrifugation, such as a sucrose gradient or a sucrose cushion. 

Adding protease inhibitors is essential to monitor protein levels and prevent protein degradation by proteases. Many protease inhibitors represent a mix of several compounds targeting various cellular proteases. However, for some specific targets, it may be necessary to include additional specific protease inhibitors. Note that some proteins only interact with individual proteases upon lysis of membranes.  

1. Place the cell culture dish on ice and wash the cells with ice-cold PBS.  
2. Aspirate the PBS, then add ice-cold lysis buffer (1 mL per 107 cells/100 mm dish/150 cm2 flask; 0.5 mL per 5x106 cells/60 mm dish/75 cm2 flask). 
3. Scrape adherent cells off the dish using a cold plastic cell scraper, then gently transfer the cell suspension into a pre-cooled microcentrifuge tube. Alternatively, cells can be trypsinized and washed with PBS before resuspension in lysis buffer in a microcentrifuge tube. 
4. Maintain constant agitation for 30 min at 4°C. 
5. Centrifuge in a microcentrifuge at 4°C. You may have to adjust the centrifugation force and time depending on the cell type. A general guideline is 20 min at 12,000 rpm, but this must be determined for your experiment (eg., leukocytes need very light centrifugation). 
6. Gently remove the tubes from the centrifuge and place them on ice. Aspirate the supernatant and transfer it to a fresh tube kept on ice while discarding the pellet.

1. Dissect the tissue of interest with clean tools, preferably on ice, and as quickly as possible to prevent protease degradation. 
2. Place the tissue in round-bottom microcentrifuge tubes or Eppendorf tubes and immerse it in liquid nitrogen to snap freeze. Store samples at -80°C for later use or keep them on ice for immediate homogenization. 
3. For a ~5 mg piece of tissue, add ~300 μL of ice-cold lysis buffer rapidly to the tube, homogenize with an electric homogenizer, rinse the blade twice with another 2 x 300 μL of lysis buffer, then maintain constant agitation for 2 hours at 4°C (eg, place your sample on an orbital shaker in the fridge). The volumes of lysis buffer must be determined depending on the amount of tissue present. Protein extract should not be too diluted to avoid protein loss and large volumes of samples to be loaded onto gels. The minimum recommended concentration is 0.1 mg/mL, while the optimal concentration is 1–5 mg/mL). 
4. Centrifuge the tubes for 20 min at 12,000 rpm at 4°C in a microcentrifuge. Gently remove the tubes from the centrifuge and place them on ice; then aspirate the supernatant and transfer it to a fresh tube kept on ice. Discard the pellet.

Once the samples are fully lysed, measure protein concentration. 

1. Perform a Bradford assay, a Lowry assay, or a bicinchoninic acid (BCA) assay. Bovine serum albumin (BSA) is a frequently used protein standard. 
2. Once you have determined the concentration of each sample, you can freeze them at -20°C or -80°C for later use or prepare them for immunoprecipitation or loading onto a gel.

To ensure proper detection of the protein of interest, it is necessary to denature it, as antibodies typically recognize a specific region called an epitope, which may be located within the protein's 3D conformation. Denaturation involves unfolding the protein to expose the epitope. 

To denature the protein, we use a loading buffer with the anionic detergent sodium dodecyl sulfate (SDS). The sample is then heated by boiling the mixture at 95–100°C for 5 minutes. Alternatively, heating at 70°C for 5–10 minutes is acceptable, especially when studying multi-pass membrane proteins that tend to aggregate when boiled, hindering efficient gel entry. 

The standard loading buffer is called 2X Laemmli buffer¹. It can also be prepared at 4X and 6X strength to minimize the dilution of the samples. The 2X Laemmli buffer is to be mixed with the sample in 1:1 ratio. For the 2x Laemmli buffer recipe, refer to Buffers and stock solutions for western blot. 

When SDS is used with proteins, it imparts a negative charge to all proteins by binding to them. SDS attaches to proteins in a mass ratio of 1.4:1, resulting in negatively charged denatured polypeptides that migrate based on their molecular weight rather than their intrinsic charge density. 

Using high-quality SDS grade is crucial for obtaining clear protein separation. A protein-stained background, along with indistinct or slightly distinct protein bands, indicates old or poor-quality SDS. Including 2-mercaptoethanol or dithiothreitol in the buffer reduces disulfide bridges, which is necessary for separation by size. 

Glycerol is added to the loading buffer to increase the sample's density, keeping it at the bottom of the well and preventing overflow and uneven gel loading. 

To visualize the protein migration, it is common to include a small anionic dye molecule in the loading buffer (eg, bromophenol blue). The dye, being anionic and small, migrates the fastest among the components in the mixture, serving as a migration front to monitor the separation progress.  

During the protein sample treatment, the sample should be mixed by vortexing before and after the heating step for the optimal resolution. After vortexing, we recommend a short centrifugation step to remove any residual liquid from the cap or tube walls. This step is especially critical when loading the entire volume. To ensure equal loading and avoid loading bubbles, we suggest to prepare a higher sample volume than necessary and not load the entire volume. 

In some cases, antibodies may recognize epitopes composed of non-contiguous amino acids. Although these amino acids are not sequential in the protein's primary sequence, they are in close proximity within the folded three-dimensional structure. Antibodies can only recognize such epitopes as they appear on the surface of the folded structure. 

In these circumstances, we must run a western blot under non-denaturing conditions, as indicated in the datasheet's applications section. In non-denaturing conditions, the sample and migration buffers do not contain sodium dodecyl sulfate (SDS), and the samples are not heated. 

Furthermore, certain antibodies only recognize proteins in their non-reduced form, particularly on cysteine residues. When using such antibodies, omit the reducing agents, such as β-mercaptoethanol and dithiothreitol (DTT), from the loading and migration buffers.

Rule of thumb: reduce and denature unless the datasheet specifies otherwise.

References:

1. Laemmli UK, 1970. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature, 227, 680–5.