Western Blot Sample Preparation
Lysis
The first step in sample preparation is isolating proteins from their source. Usually, proteins are isolated from cells or tissues via lysis. Lysis breaks down the cell membrane to separate proteins from the non-soluble parts of the cell. A number of lysis buffers can be used to prepare samples for western blotting. In general, these buffers vary in the strength of their detergents to release soluble proteins. Stronger detergents, such as Triton X-100 are recommended for difficult to solubilize proteins. Refer to the list below for common lysis buffer recipes.
The sub-cellular localization of the protein of interest can be used as a starting point to determine the optimal lysis buffer to obtain high protein purity and yield. Proteins found predominantly or exclusively in a sub-cellular location, such as nucleus or mitochondria, can be enriched with fractionation kits. This is particularly useful when probing weakly expressed proteins. Since each protein is different, lysis buffer and detergent conditions may require optimization for individual western blotting experiments. Refer to the table below as a starting point to choose a lysis buffer.
Lysis Buffer Recommendations
| Cell Location | Recommended Buffer |
|---|---|
| Whole Cell Lysate | NP-40 |
| Nucleus | RIPA |
| Mitochondria | RIPA |
| Cytoplasm | Tris-HCl |
| Membrane-bound Protein |
RIPA (SDS is generally considered harsh and thus is often well-suited for difficult to solubilize proteins) |
Common Lysis Buffer Recipes
| Buffer | Components |
|---|---|
| NP-40 | 150 mM NaCl 1% NP-40 or Triton X-100 50 mM Tris pH 8.0 |
| RIPA | 150 mM NaCl 1% NP-40 or Triton X-100 0.5% Sodium deoxycholate 0.1% SDS 50 mM Tris, pH 8.0 |
| Tris-HCl | 20 mM Tris-HCl, pH 7.5 |
Protease and Phosphatase Inhibitors
Immediately following cell lysis, proteolysis, dephosphorylation, and denaturation begin to occur. This activity should be kept to a minimum by preparing samples on ice or at 4˚C and by adding protease and phosphatase inhibitors fresh to the lysis buffer.
While there are many commercially available ready-to-use inhibitor cocktails (often proprietary), a homemade mix can be made based on individual needs. The table below lists common protease and phosphatase inhibitors, their targets, and the recommended final concentration in the lysis buffer.
Common Protease and Phosphatase Inhibitors
| Inhibitor | Target | Final Concentration |
|---|---|---|
| Aprotinin (Catalog # 4139) | Trypsin, chymotrypsin, plasmin | 2 µg/mL |
| Leupeptin (Catalog # 1167) | Lysosomal | 1-10 µg/mL |
| Pepstatin A (Catalog # 1190) | Aspartic proteases | 1 µg/mL |
| PMSF (Catalog # 4486) | Serine proteases | 1 mM |
| EDTA (Catalog # 4250-050-04) | Mg2+ and Mn2+ metalloproteases | 1-5 mM |
| EGTA (Catalog # 2807) | Ca2+ metalloproteases | 1 mM |
| Sodium fluoride | Serine & threonine phosphatases | 5-10 mM |
| Orthovanadate (Catalog # 2821) | Tyrosine phosphatases | 1 mM |
| Pyrophosphate | Serine & threonine phosphatases | 1-2 mM |
| B-glycerophosphate | Serine & threonine phosphatases | 1-2 mM |
Carrying Out Lysis
The cell lysis protocol can vary widely depending on the cell or tissue sample used. For example, lysing heart or brain tissue from a mouse may require homogenization, which typically involves flash freezing the sample in liquid nitrogen prior to grinding with a mortar and pestle or an electric homogenizer. A lysis protocol should be chosen based on standard protocols in the field for the given tissue. The following is a general example of a protocol for lysing cells grown in culture.
Lysate Preparation from Adherent Cell Culture
- Wash cell culture dish on ice with ice-cold PBS.
- Aspirate PBS and add ice-cold lysis buffer (1 mL per confluent 107 cells/100mm dish/150 cm2 flask). See the table above for lysis buffer recommendations based on the subcellular location of the protein of interest.
- Using a cell scrapper, scrape adherent cells off the dish and transfer the cell suspension into a microcentrifuge tube. If required, the cells can be trypsinized and washed with PBS prior to resuspension in lysis buffer.
- Agitate cells for 30 minutes at 4˚C
- Centrifuge cell lysate mixture at 4˚C.
Note: The time and centrifugation force vary for each cell type, but a general guideline is 20 minutes at 12,000 rpm. - Transfer the supernatant (lysate) to a fresh tube on ice.
Determining Lysate Protein Concentration
It is important to determine the protein concentration within each lysate in order to ensure equal loading of the SDS-PAGE gel in subsequent steps. Equal loading allows proteins levels and expression differences to be accurately quantified in western blotting. Protein concentration can be determined by performing a standard Bradford, Lowry, or BCA assay. Protein samples can be frozen at -20 ˚C or -80 ˚C for later use or prepared for gel loading for immediate use.
Preparing Lysates for Gel Loading
The epitope usually resides within the 3D conformation of the protein. Thus, it is necessary to unfold or denature the protein to enable access to the antibody. Denaturing is performed by briefly boiling the sample in a loading buffer containing SDS.
The most common loading buffer for SDS-PAGE gel electrophoresis is 2X Laemmli buffer. It can also be made at 4X or 6X concentration, which may be helpful if larger volumes of lysates with low protein concentration.
Loading Buffer Recipe
| Loading Buffer | Components |
|---|---|
| 2X Laemmli buffer | 4% SDS 5% 2-mercaptoethanol 20% glycerol 0.004% bromophenol blue 0.125 M Tris HCl Adjust pH to 6.8 |
Understanding the Loading Buffer Components:
| Component | Notes |
|---|---|
| SDS | Causes proteins to become negatively charged by their attachment to SDS anions. The SDS wrapping around the polypeptide backbone causes protein denaturation. The negative charged conferred by SDS to polypeptide chains is proportional to their length. Because of this, proteins can be separated by SDS-PAGE electrophoresis according to their molecular weight and not by intrinsic electrical charge. Both the loading buffer and the gel running buffer contain SDS to allow this. |
| Reducing agents: β-ME or DTT | While SDS serves to unfold proteins, reducing agents further remove tertiary and quaternary structure by reducing intramolecular and intermolecular disulfide bonds, converting proteins to a linear form. |
| Glycerol | Is added to sample loading buffer to increase sample density so, when samples are loaded into the gel, they sink to the bottom of the well. This promotes even protein loading between wells and minimizing sample overflow. |
| Bromophenol blue | Bromophenol blue is a small anionic dye that is added to enable the visualization of protein migration during gel electrophoresis. Because of its small size, bromophenol blue migrates faster than the proteins in the samples and provides a migration front to monitor the electrophoresis process and prevent sample run-off. |
Loading and Running Buffer Conditions
| Protein State | Sample Loading Buffer | Gel Running Buffer |
|---|---|---|
| Reduced and denatured (most common) | SDS + β-ME or DTT | SDS |
| Reduced and native | β-ME or DTT, No SDS | No SDS |
| Oxidized and denatured | SDS, No β-ME or DTT | SDS |
| Oxidized and native | No SDS and No β-ME or DTT | No SDS |
Sample Preparation for Gel Loading
- Determine the protein concentration of each cell lysate.
- Determine how much protein to load (Recommended: 10-50 μg/lane) and add an equal volume of 2X Laemmli buffer.
- Reduce and denature the samples by boiling the lysates in Laemmli Buffer at 95-100˚C for 5 minutes.
Note: This step should only be skipped if the antibody datasheet recommends non-reducing or non-denaturing conditions. Occasionally, antibodies only recognize epitopes as they exist on the surface of a protein’s native, non-denatured state. Non-denaturing conditions can be generated by leaving SDS out of the sample and migration buffers and not boiling the samples. Further, certain antibodies only recognize proteins in their non-reduced, or oxidized forms. In this case, the reducing-agents β-mercaptoethanol (βME) or DTT should not be included in the buffers. Refer to the table above for loading buffer and gel running buffer guidelines.